Patent application title: PRO-CLOTTING ENZYME, AND METHOD FOR DETECTION OF ENDOTOXIN OR (1-3)-BETA-D-GLUCAN USING THE SAME
Inventors:
Hiroshi Tamura (Tokyo, JP)
Hiroshi Tamura (Tokyo, JP)
Shoji Takahashi (Tokyo, JP)
Assignees:
SEIKAGAKU CORPORATION
IPC8 Class: AC12Q126FI
USPC Class:
435 25
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving oxidoreductase
Publication date: 2009-08-20
Patent application number: 20090208995
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Patent application title: PRO-CLOTTING ENZYME, AND METHOD FOR DETECTION OF ENDOTOXIN OR (1-3)-BETA-D-GLUCAN USING THE SAME
Inventors:
Hiroshi Tamura
Shoji Takahashi
Agents:
SUGHRUE MION, PLLC
Assignees:
SEIKAGAKU CORPORATION
Origin: WASHINGTON, DC US
IPC8 Class: AC12Q126FI
USPC Class:
435 25
Abstract:
Objects of the present invention are to provide a DNA fragment encoding a
limulus-derived pro-clotting enzyme, a virus harboring the DNA fragment,
a cell harboring the virus, a method of producing the pro-clotting enzyme
by use of the cell, and means for assaying an endotoxin or
(1→3)-β-D-glucan employing the enzyme, wherein these elements
are capable of producing an endotoxin or (1→3)-β-D-glucan
assay reagent of satisfactory quality, steadily, at low cost, and on a
large scale. In the present invention, for example, a DNA fragment
encoding a protein having an amino acid sequence defined by SEQ ID NO: 4
is selected as a nucleic acid fragment encoding a limulus-derived
pro-clotting enzyme, and the corresponding recombinant pro-clotting
enzyme. Use of the enzyme can provide a high-sensitivity method and kit
for detecting (1→3)-β-D-glucan and an endotoxin, utilizing a
cascade reaction system in a horseshoe crab lysate.Claims:
1. A nucleic acid fragment encoding a pro-clotting enzyme derived from a
horseshoe crab.
2. The nucleic acid fragment according to claim 1, wherein the horseshoe crab is selected from among Tachypleus tridentatus, Limulus polyphemus, Tachypleus gigas, and Tachypleus rotundicauda.
3. The nucleic acid fragment according to claim 1, wherein the nucleic acid fragment encoding a pro-clotting enzyme derived from a horseshoe crab is selected from the following nucleic acid fragments (A) to (C):(A) a DNA fragment encoding a protein having an amino acid sequence defined by SEQ ID NO: 4,(B) a DNA fragment encoding a protein having an amino acid sequence defined by SEQ ID NO: 4 in which one or several amino acid residues are deleted, substituted, inserted, or translocated, and having activity of a pro-clotting enzyme derived from a horseshoe crab, and(C) an RNA fragment produced through transcription of the DNA fragment (A) or (B).
4. The nucleic acid fragment according to claim 1, wherein the nucleic acid fragment encoding a pro-clotting enzyme derived from a horseshoe crab is selected from the following nucleic acid fragments (a) to (c):(a) a DNA fragment having a nucleotide sequence defined by nucleotides 1 to 1143 in SEQ ID NO: 3,(b) a DNA fragment having a nucleotide mutation in a nucleotide sequence containing a nucleotide sequence defined by nucleotides 1 to 1143 in SEQ ID NO: 3, wherein the mutation causes deletion, substitution, insertion, or translocation of one or several amino acid residues in an amino acid sequence of a protein encoded by the nucleotide sequence defined by nucleotides 1 to 1143 in SEQ ID NO: 3, and a protein expressed by the DNA fragment having a nucleotide mutation has activity of a pro-clotting enzyme derived from a horseshoe crab, and(c) an RNA fragment produced through transcription of the DNA fragment (a) or (b).
5. A virus harboring a nucleic acid fragment according to claim 1.
6. The virus according to claim 5, wherein the virus is baculovirus.
7. The virus according to claim 6, wherein the baculovirus is nuclear polyhedrosis virus.
8. A cell harboring a virus according to claim 5.
9. The cell according to claim 8, which is a cell derived from an insect.
10. A method of producing a pro-clotting enzyme derived from a horseshoe crab, the method comprising the steps of growing a cell according to claim 8, and preparing the pro-clotting enzyme from the growth product.
11. A pro-clotting enzyme produced through a method according to claim 10.
12. A method of detecting an endotoxin, wherein the method comprises:causing a serine protease precursor to be co-present with a pro-clotting enzyme according to claim 11 in an endotoxin-detection sample, said serine protease precursor expressing an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with the endotoxin; anddetecting the endotoxin present in the sample through employing, as an index, an enzymatic activity in conversion of a pro-clotting enzyme to a clotting enzyme.
13. The method of detecting an endotoxin according to claim 12, wherein the serine protease precursor which expresses an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with the endotoxin comprises a serine protease precursor which expresses an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with activated factor C, and factor C derived from a horseshoe crab and/or recombinant factor C.
14. The method of detecting an endotoxin according to claim 13, wherein the serine protease precursor which expresses an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with activated factor C is factor B derived from a horseshoe crab and/or recombinant factor B.
15. A method of detecting an endotoxin, wherein the method comprises:causing a serine protease precursor to be co-present with a pro-clotting enzyme according to claim 11 in an endotoxin-detection sample, said serine protease precursor expressing an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with the endotoxin; anddetecting the endotoxin present in the sample through employing, as an index, an enzymatic activity in conversion of a pro-clotting enzyme to a clotting enzyme,wherein a pro-clotting enzyme according to claim 11, recombinant factor C, and recombinant factor B are exclusively caused to be co-present as cascade reaction proteins.
16. An endotoxin-detection kit for carrying out a detection method, wherein the method comprises:causing a serine protease precursor to be co-present with a pro-clotting enzyme according to claim 11 in an endotoxin-detection sample, said serine protease precursor expressing an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with the endotoxin; anddetecting the endotoxin present in the sample through employing, as an index, an enzymatic activity in conversion of a pro-clotting enzyme to a clotting enzyme,wherein the kit comprises, as essential components, a pro-clotting enzyme according to claim 11, and a serine protease precursor which expresses an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with the endotoxin.
17. The endotoxin-detection kit according to claim 16, wherein the serine protease precursor which expresses an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with the endotoxin comprises a serine protease precursor which expresses an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with activated factor C, and factor C derived from a horseshoe crab and/or recombinant factor C.
18. The endotoxin-detection kit according to claim 17, wherein the serine protease precursor which expresses an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with activated factor C is factor B derived from a horseshoe crab and/or recombinant factor B.
19. An endotoxin-detection kit for carrying out a detection method, wherein the method comprises:causing a serine protease precursor to be co-present with a pro-clotting enzyme according to claim 11 in an endotoxin-detection sample, said serine protease precursor expressing an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with the endotoxin; anddetecting the endotoxin present in the sample through employing, as an index, an enzymatic activity in conversion of a pro-clotting enzyme to a clotting enzyme,wherein the kit comprises, as essential components, a pro-clotting enzyme according to claim 11, and a serine protease precursor which expresses an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with the endotoxin,wherein the kit contains, as cascade reaction proteins, exclusively a pro-clotting enzyme according to claim 11, recombinant factor C, and recombinant factor B.
20. A method of detecting (1.fwdarw.3)-.beta.-D-glucan, wherein the method comprises:causing a serine protease precursor to be co-present with a pro-clotting enzyme according to claim 11 in a (1.fwdarw.3)-.beta.-D-glucan-detection sample, said serine protease precursor expressing an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with (1.fwdarw.3)-.beta.-D-glucan; anddetecting (1.fwdarw.3)-.beta.-D-glucan present in the sample through employing, as an index, enzymatic activity in conversion of a pro-clotting enzyme to a clotting enzyme.
21. The method of detecting (1.fwdarw.3)-.beta.-D-glucan according to claim 20, wherein the serine protease precursor which expresses an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with (1.fwdarw.3)-.beta.-D-glucan comprises factor G derived from a horseshoe crab and/or recombinant factor G.
22. A method of detecting (1.fwdarw.3)-.beta.-D-glucan, wherein the method comprises:causing a serine protease precursor to be co-present with a pro-clotting enzyme according to claim 11 in a (1.fwdarw.3)-.beta.-D-glucan-detection sample, said serine protease precursor expressing an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with (1.fwdarw.3)-.beta.-D-glucan; anddetecting (1.fwdarw.3)-.beta.-D-glucan present in the sample through employing, as an index, enzymatic activity in conversion of a pro-clotting enzyme to a clotting enzyme,wherein a pro-clotting enzyme according to claim 11 and recombinant factor G are exclusively caused to be co-present as cascade reaction proteins.
23. A (1.fwdarw.3)-.beta.-D-glucan-detection kit for carrying out a detection method, wherein the method comprises:causing a serine protease precursor to be co-present with a pro-clotting enzyme according to claim 11 in a (1.fwdarw.3)-.beta.-D-glucan-detection sample, said serine protease precursor expressing an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with (1.fwdarw.3)-.beta.-D-glucan; anddetecting (1.fwdarw.3)-.beta.-D-glucan present in the sample through employing, as an index, enzymatic activity in conversion of a pro-clotting enzyme to a clotting enzyme,comprising, as essential components, a pro-clotting enzyme according to claim 11, and a serine protease precursor which expresses an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with (1.fwdarw.3)-.beta.-D-glucan.
24. The (1.fwdarw.3)-.beta.-D-glucan-detection kit according to claim 23, wherein the serine protease precursor which expresses an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with (1.fwdarw.3)-.beta.-D-glucan comprises factor G derived from a horseshoe crab and/or recombinant factor G.
25. A (1.fwdarw.3)-.beta.-D-glucan-detection kit for carrying out a detection method, wherein the method comprises:causing a serine protease precursor to be co-present with a pro-clotting enzyme according to claim 11 in a (1.fwdarw.3)-.beta.-D-glucan-detection sample, said serine protease precursor expressing an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with (1.fwdarw.3)-.beta.-D-glucan; anddetecting (1.fwdarw.3)-.beta.-D-glucan present in the sample through employing, as an index, enzymatic activity in conversion of a pro-clotting enzyme to a clotting enzyme,the kit comprising, as essential components, a pro-clotting enzyme according to claim 11, and a serine protease precursor which expresses an activity of converting a pro-clotting enzyme to a clotting enzyme upon contact with (1.fwdarw.3)-.beta.-D-glucan,wherein the kit contains, as cascade reaction proteins, exclusively a pro-clotting enzyme according to claim 11 and recombinant factor G.
Description:
TECHNICAL FIELD
[0001]The present invention relates to a nucleic acid fragment encoding a pro-clotting enzyme derived from a horseshoe crab (hereinafter may be referred to as a limulus-derived pro-clotting enzyme), to a virus harboring the nucleic acid fragment, to a cell harboring the virus, to a method of producing the pro-clotting enzyme by use of the cell, and to a method and a kit for detecting Et or BG employing the pro-clotting enzyme produced through the production method.
BACKGROUND ART
[0002]There are disclosed methods for determining Et or BG by use of an amebocyte lysate of a horseshoe crab (i.e., a horseshoe crab hematocyte extract, hereinafter referred to simply as a lysate). These methods are based on coagulation of the lysate by Et or BG. The coagulation reaction occurs through cascade reaction of several coagulation factors (Patent Document 1 and Non-Patent Document 1).
[0003]For example, when BG is brought into contact with the lysate, factor G contained in the lysate is activated, to thereby form activated factor G. The activated factor G activates Pro-CE present in the lysate, to thereby form CE. CE hydrolyzes a specific site of a coagulogen molecule present in the lysate, thereby forming coaguline gel, leading to coagulation of the lysate. CE also acts on a synthetic substrate (e.g., t-butoxycarbonyl-leucyl-glycyl-arginine-pNA (Boc-Leu-Gly-Arg-pNA)), to thereby hydrolyze the amino bonds, whereby pNA is released. Thus, BG can be determined through measuring absorbance of the formed coloring substance (pNA) (Patent Document 1).
[0004]Although Pro-CE was previously cloned (Non-Patent Document 2), a protein (Pro-CE) maintaining an enzymatic activity is difficult to develop when the corresponding nucleic acid fragment is employed.
[0005]In other words, although Pro-CE was previously cloned, the technique and the resultant protein disclosed in Non-Patent Document 2 are merely included in a standard technique for producing a target clone of a cDNA fragment encoding a protein of interest. More specifically, the standard technique is limited to selection of 23 clones from a λgt11 cDNA library (1,500,000 clones) by use of an anti-CE antibody, subcloning to a pUC118/119 vector, and determination of the nucleotide sequence. In fact, these working steps are considerably heavy, and there have not yet been performed development of an enzymatic activity of Pro-CE, which is a precursor of serine protease [protease (amidase) activity of CE], and activation of Pro-CE by activated factor B or quantitative determination (including reproduction test) of the enzymatic activity of Pro-CE in the co-presence of activated factor B or C. Completely differing from determination of the nucleotide sequence of a specific protein, yielding the specific protein as a recombinant and establishing a specific assay system employing the recombinant protein can be attained only by highly advanced creation of technical ideas.
Patent Document 1: Japanese Patent Application laid-Open (kokai) No. 08-122334Patent Document 2: Japanese Patent Application laid-Open (kokai) No. 2006-271384Non-Patent document 1: J. Protein Chem., 5, p. 255-268 (1986)Non-Patent document 2: J. Biol. Chem., 265(36), p. 22426-22433 (1990)
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0006]Thus, objects of the present invention are to provide a nucleic acid fragment encoding a limulus-derived Pro-CE, a virus harboring the nucleic acid fragment, a cell harboring the virus, a method of producing the Pro-CE by use of the cell, and a method and a kit for detecting Et or BG employing the Pro-CE produced through the production method, wherein these elements are capable of mass-producing an Et or BG assay reagent of satisfactory quality, steadily, at low cost, and on a large scale.
[0007]The present inventors have conducted extensive studies in order to attain the aforementioned objects, and have found that a protein having Pro-CE activity can be produced by use of a cell harboring a virus containing a DNA fragment encoding Pro-CE, whereby an Et or BG assay reagent of satisfactory quality can be mass-produced steadily, at low cost, and on a large scale. The present invention has been accomplished on the basis of this finding.
[0008]Abbreviations used in the present invention (including the aforementioned "Background") are as follows.
Pro-CE: pro-clotting enzymeCE: clotting enzymeAcNPV: nuclear polyhedrosis virus of Autographa californica
BG: (1→3)-β-D-glucan
[0009]Et: endotoxin (also referred to as lipopolysaccharide)HEPES: 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acidHRP: horseradish peroxidaseMOI: multiplicity of infectionNPV: nuclear polyhedrosis virusPBS: phosphate buffered salinePCR: polymerase chain reactionpNA: p-nitroanilinePVDF: polyvinylidene difluorideSDS: sodium dodecyl sulfateSDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresisLAL: limulus amebocyte lysate
[0010]Accordingly, the present invention provides a nucleic acid fragment encoding a limulus-derived Pro-CE (hereinafter the nucleic acid fragment may be referred to as a "nucleic acid fragment of the present invention").
[0011]The horseshoe crab (limulus) employed in the present invention is selected from among the following four species: Tachypleus tridentatus, Limulus polyphemus, Tachypleus gigas, and Tachypleus rotundicauda (Carcinoscorpius rotundicauda).
[0012]The "nucleic acid fragment encoding a limulus-derived Pro-CE" employed in the invention is preferably selected from the following nucleic acid fragments (A) to (C):
[0013](A) a DNA fragment encoding a protein having an amino acid sequence defined by SEQ ID NO: 4,
[0014](B) a DNA fragment encoding a protein having an amino acid sequence defined by SEQ ID NO: 4 in which one or several amino acid residues are deleted, substituted, inserted, or translocated, and having activity of a limulus-derived Pro-CE, and
[0015](C) an RNA fragment produced through transcription of the DNA fragment (A) or (B).
[0016]The "nucleic acid fragment encoding a limulus-derived Pro-CE" employed in the invention is preferably selected from the following nucleic acid fragments (a) to (c):
[0017](a) a DNA fragment having a nucleotide sequence defined by nucleotides 1 to 1143 in SEQ ID NO: 3,
[0018](b) a DNA fragment having a nucleotide mutation in a nucleotide sequence containing a nucleotide sequence defined by nucleotides 1 to 1143 in SEQ ID NO: 3, wherein the mutation causes deletion, substitution, insertion, or translocation of one or several amino acid residues in an amino acid sequence of a protein encoded by the nucleotide sequence defined by nucleotides 1 to 1143 in SEQ ID NO: 3, and a protein expressed by the DNA fragment having a nucleotide mutation has activity of a limulus-derived Pro-CE, and
[0019](c) an RNA fragment produced through transcription of the DNA fragment (a) or (b).
[0020]The present invention also provides a virus harboring the nucleic acid fragment of the present invention (hereinafter referred to as a "virus of the present invention").
[0021]The "virus" employed in the invention is preferably a baculovirus. Among baculoviruses, NPV is preferred, with AcNPV being more preferred.
[0022]The present invention also provides a cell harboring the virus of the present invention (hereinafter referred to as a "cell of the present invention").
[0023]No particular limitation is imposed on the "cell," and the cell is freely selected in consideration of, for example, matching with the virus of the present invention. Examples of the cell include cells derived from E. coli, bacteria, yeasts, and insects. As mentioned above, the virus of the present invention is preferably a baculovirus. When a baculovirus is employed, an insect-derived cell is preferably selected.
[0024]The present invention also provides a method of producing a limulus-derived Pro-CE, the method comprising the steps of growing the cell of the present invention, and preparing a limulus-derived Pro-CE from the growth product (hereinafter referred to as a "production method of the present invention").
[0025]The present invention also provides a limulus-derived Pro-CE produced through the production method of the present invention (hereinafter referred to as an "enzyme of the present invention").
[0026]The present invention also provides a method of detecting Et, wherein the method comprises:
[0027]causing "a serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with Et" to be co-present with the enzyme of the present invention in a test sample in which the Et possibly present therein is to be detected (hereinafter may be referred to as an "Et-detection sample"); and
[0028]detecting Et present in the sample through employing, as an index, enzymatic activity in conversion of Pro-CE to CE (hereinafter referred to as a "method 1 of the present invention").
[0029]In the method 1 of the present invention, the "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with the Et" preferably comprises a "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with activated factor C", and "limulus-derived factor C and/or recombinant factor C."
[0030]The "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with activated factor C" is preferably limulus-derived factor B and/or recombinant factor B.
[0031]The aforementioned factors C and B are preferably recombinants. In one best mode of the method 1 of the present invention, the enzyme of the present invention is caused to be co-present with factors C and B in an Et-detection test sample, and the employed factors C and B are recombinants. Carrying out the method 1 of the present invention by use of limulus-derived native factors C and B are considerably preferred from the viewpoint of effectiveness of method, such as detection sensitivity. However, in order to collect the hemolymph of horseshoe crab serving as a raw material for native factors C and B, horseshoe crabs must be captured, and their blood must be collected in such an amount that continual growth thereof is not impaired. In addition, a certain level of stress is unavoidably imposed on horseshoe crabs upon capturing and blood collection, and some people point out a decrease in the number of horseshoe crabs caused by environmental destruction, etc. Thus, it is deeply significant that recombinant proteins are employed as all proteins involved in cascade reaction for carrying out the method 1 of the present invention, particularly from the viewpoint of protection of precious biological resources. Use of recombinant proteins greatly contributes to the protection of lifeforms and provision of animal alternatives.
[0032]In the case where recombinant factors C and B are employed as essential elements of the Et-detection system, no particular limitation is imposed on the selection of a vector into which a gene encoding the corresponding factor is introduced and on the host to which the vector is transduced. However, similar to the case of the aforementioned recombinant Pro-CE, the virus serving as a vector is preferably a baculovirus. Among baculoviruses, NPV is preferred, with AcNPV being more preferred. Examples of the host include cells derived from E. coli, bacteria, yeasts, and insects. As mentioned above, the virus of the present invention is preferably a baculovirus. When a baculovirus is employed, an insect-derived cell is preferably selected.
[0033]The concept "employing, as an index, an enzymatic activity in conversion of the enzyme of the present invention to CE" means that the enzymatic activity induced through conversion of Pro-CE (the enzyme of the present invention) to CE is detected quantitatively or qualitatively, whereby the detected enzymatic activity is employed as an index for the amount or presence of Et in the test sample. Examples of the "phenomenon that exhibits enzymatic activity induced through conversion of Pro-CE to CE" include coagulation of horseshoe crab amebocyte lysate through formation of coaguline gel in the presence of CE (which can be detected through observation of gelation, change in turbidity, etc.) and developing color following amide bond cleavage of a synthetic chromogenic substrate. Particularly when the synthetic chromogenic substrate is employed, an assay system of high sensitivity and good reproducibility can be established. Such an assay system, which does not necessarily employ a limulus-derived lysate, is very advantageous from the viewpoint of protection of precious biological resources.
[0034]Examples of the synthetic chromogenic substrate include X-A-Y (wherein X represents a protective group, Y represents a coloring dye, and A represents a tripeptide). The A-Y bond is cleaved in the presence of CE, to thereby cause the coloring dye Y to develop color, which serves as an index for quantitatively or qualitatively detect an Et. Examples of the protective group X include known peptide protective groups such as a t-butoxycarbonyl group and a benzoyl group. Examples of the coloring dye Y include pNA, MCA (7-methoxycoumarin-4-acetic acid), DNP (2,4-dinitroaniline), and Dansyl dye. Examples of the tripeptide include Leu-Gly-Arg (LGR), Ile-Glu-Gly-Arg (IEGR), and Val-Pro-Arg (VPR).
[0035]For quantitatively carrying out the method 1 of the present invention, a correlation (typically a calibration curve) between the Et levels determined by use of a standard Et and the corresponding index intensities (absorbance (optical density) for coloring dye Y, turbidity attributed to coagulation of lysate, etc.) is established in advance. The Et level of a test sample can be quantitatively determined from an actually detected index intensity on the basis of the established correlation.
[0036]No particular limitation is imposed on the test sample, and examples of the sample include injection water, pharmaceuticals, infusions, blood preparations, medical apparatus (medical tools), quasi-drugs, cosmetics, foods, beverages, environmental samples (air, river water, soil, etc.), native proteins, genetically modified proteins, nucleic acids, enzymes, saccharides, electrolytes, and bio-samples (blood, body fluid, tissue, etc.).
[0037]The present invention also provides an Et-detection kit for carrying out the method 1 of the present invention (hereinafter referred to as a "kit 1 of the present invention"), wherein the kit comprises, as essential components, the enzyme of the present invention and a "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with Et."
[0038]In the kit 1 of the present invention, the "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with Et" is preferably a "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with activated factor C", and "limulus-derived factor C and/or recombinant factor C."
[0039]The "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with activated factor C" is preferably limulus-derived factor B and/or recombinant factor B.
[0040]In the kit 1 of the present invention, the aforementioned factors C and B are preferably recombinants. In one best mode of the kit 1 of the present invention, the employed factors C and B are recombinants. In other words, the proteins contained in the kit 1 of the present invention and involved in cascade reaction more preferably include only the enzyme of the present invention and recombinant factors C and B.
[0041]The kit 1 of the present invention may also include reagents for carrying out the method 1 of the present invention; e.g., the aforementioned synthetic chromogenic substrate (X-A-Y), buffer, diluent, salt, and limulus-derived amebocyte lysate, selected in accordance with the mode of the method 1 of the present invention employing the kit 1 of the present invention.
[0042]The present invention also provides a method of detecting BG, wherein the method comprises:
[0043]causing a "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with BG" to be co-present with the enzyme of the present invention in a test sample in which BG possibly present therein is to be detected (hereinafter may be referred to as a "BG-detection sample"); and
[0044]detecting BG present in the sample through employing, as an index, enzymatic activity in conversion of Pro-CE to CE (hereinafter the method referred to as a "method 2 of the present invention").
[0045]In the method 2 of the present invention, the "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with BG" is preferably limulus-derived factor G and/or recombinant factor G.
[0046]The aforementioned factor G is preferably recombinant. In one best mode of the method 2 of the present invention, the enzyme of the present invention is caused to be co-present with factor G in a BG-detection sample, and the factor G is recombinant.
[0047]The reason why recombinant factor G is preferred in the method 2 of the present invention is the same as mentioned in relation to that recombinant factors C and B are preferred in the method 1 of the present invention. For producing recombinant factor G, the virus serving as a vector is preferably a baculovirus. Among baculoviruses, NPV is preferred, with AcNPV being more preferred. This feature is the same as described in relation to factors C and B. Examples of the host include cells derived from E. coli, bacteria, yeasts, and insects. As mentioned above, the virus of the present invention is preferably a baculovirus. When a baculovirus is employed, an insect-derived cell is preferably selected. This feature is also the same as described in relation to factors C and B.
[0048]Similar to the method 1 of the present invention, the concept "employing, as an index, an enzymatic activity in conversion of the enzyme of the present invention to CE" means that the phenomenon of conversion of Pro-CE (the enzyme of the present invention) to CE is quantitatively or qualitatively detected, whereby the detected enzymatic activity is employed as an index for the amount or presence of BG in the test sample. Examples of the phenomenon that exhibits enzymatic activity induced through conversion of Pro-CE to CE are the same as disclosed in relation to the method 1 of the present invention. The synthetic coloring substrate (X-A-Y) may also be employed in the method 2 of the present invention.
[0049]For quantitatively carrying out the method 2 of the present invention, a correlation (typically a calibration curve) between the BG levels determined by use of standard BG and the corresponding index intensities (absorbance (optical density) for coloring dye Y, turbidity attributed to coagulation of lysate, etc.) is established in advance. The BG level of a test sample can be quantitatively determined from an actually detected index intensity on the basis of the established correlation.
[0050]No particular limitation is imposed on the test sample, and examples of the sample include injection water, pharmaceuticals, infusions, blood preparations, medical apparatus (medical tools), quasi-drugs, cosmetics, foods, beverages, environmental samples (air, river water, soil, etc.), native proteins, genetically modified proteins, nucleic acids, enzymes, saccharides, electrolytes, and bio-samples (blood, body fluid, tissue, etc.).
[0051]The present invention also provides a BG-detection kit for carrying out the method 2 of the present invention (hereinafter referred to as a "kit 2 of the present invention"), wherein the kit comprises, as essential components, the enzyme of the present invention and a "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with BG."
[0052]In the kit 2 of the present invention, the "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with BG" is preferably limulus-derived factor G and/or recombinant factor G.
[0053]In the kit 2 of the present invention, the aforementioned factor G is preferably a recombinant. In one best mode of the kit 2 of the present invention, the employed recombinant factor G includes entire factor G molecular components (α- and β-subunits). In other words, the proteins contained in the kit 2 of the present invention and involved in cascade reaction preferably include exclusively the enzyme of the present invention and recombinant factor G.
[0054]The kit 2 of the present invention may also include reagents for carrying out the method 2 of the present invention; e.g., the aforementioned synthetic chromogenic substrate (X-A-Y), buffer, diluent, salt, and limulus-derived lysate, selected in accordance with the mode of the method 2 of the present invention employing the kit 2 of the present invention.
EFFECTS OF THE INVENTION
[0055]The nucleic acid fragment of the present invention is very useful, since the virus of the present invention, which is useful for steady, low-cost, and large-scale production of Pro-CE of satisfactory quality, can be provided through employment of the nucleic acid fragment. The virus of the present invention is very useful, since the cell of the present invention, which is useful for steady, low-cost, and large-scale production of Pro-CE of satisfactory quality, can be provided through employment of the virus. The cell of the present invention is very useful, since the cell can produce a protein maintaining Pro-CE activity and satisfactory quality, steadily, at low cost, and on a large scale and can also provide the method and kit of the present invention. The method and kit of the present invention realize detection and measurement of Et and BG without preparing a lysate from horseshoe crabs, which are precious bio-resources, and can provide a very useful technique in terms of protection of lifeforms, provision of animal alternatives, cost, precision, reproducibility, etc.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056][FIG. 1] Results of nucleotide sequence analysis of the N-terminus and C-terminus regions of a target sequence of Pro-CE recombinant virus
[0057][FIG. 2] Results of western blotting showing Pro-CE expressed products.
[0058][FIG. 3] A graph showing reactivity value of supernatant fractions at a BG concentration of 1 ng/mL.
[0059][FIG. 4] A graph showing reactivity value of supernatant fractions at a BG concentration of 10 ng/mL.
[0060][FIG. 5] Reactivity value of BG in DS-3GII fraction.
[0061][FIG. 6] A graph showing reactivity value of supernatant fractions at an Et concentration of 10 ng/mL.
[0062][FIG. 7] A graph showing reactivity value of supernatant fractions at an Et concentration of 100 ng/mL.
[0063][FIG. 8] Reactivity value of BG in recombinant factor G (5-fold diluted).
[0064][FIG. 9] A graph showing the investigation results of recombinant factor B to be employed in Et reaction.
[0065][FIG. 10] A graph showing Et reactivity value of samples.
[0066][FIG. 11] A graph showing hydrolysis performance of factors to a Boc-LGR-pNA substrate in the presence of Et at high concentration.
[0067][FIG. 12] A graph showing the effect of magnesium sulfate concentration (0 to 100 mM) on Et reaction in a complete reconstitution system.
[0068][FIG. 13] A graph showing the effect of magnesium sulfate concentration (0 to 10 mM) on Et reaction in a complete reconstitution system.
[0069][FIG. 14] A graph showing the effect of calcium chloride concentration (0 to 5 mM) on Et reaction in a complete reconstitution system.
[0070][FIG. 15] A graph showing the effect of sodium chloride concentration (0 to 2.5 M) on Et reaction in a complete reconstitution system.
[0071][FIG. 16] A graph showing difference in Et reactivity value in a complete reconstitution system investigated through a test employing Boc-VPR-pNA or Boc-LGR-pNA as a substrate.
BEST MODES FOR CARRYING OUT THE INVENTION
[0072]Best modes for carrying out the present invention will next be described.
<1> The Nucleic Acid Fragment of the Present Invention
[0073]The nucleic acid fragment of the present invention is a nucleic acid fragment encoding a limulus-derived Pro-CE. No particular limitation is imposed on the "nucleic acid fragment encoding a limulus-derived Pro-CE" (i.e., the nucleic acid fragment of the present invention), so long as the nucleic acid fragment encodes a limulus-derived Pro-CE. For example, the nucleic acid fragment may be a nucleic acid fragment having a nucleotide sequence defined by SEQ ID NO: 1 (SEQ ID NO: 2 shows only the amino acid sequence corresponding to the nucleotide sequence). It will be readily appreciated by those skilled in the art that the nucleic acid fragment of the present invention also encompasses nucleic acid fragments having different nucleotide sequences attributed to degeneration of genetic codes.
[0074]The "nucleic acid fragment" employed in the present invention may be a DNA fragment or an RNA fragment, which may be selected by those skilled in the art in consideration of the use of the nucleic acid fragment. For example, when stability is emphasized, a DNA fragment may be selected.
[0075]The nucleic acid fragment of the present invention may be, for example, a nucleic acid fragment encoding Pro-CE derived from any of the following horseshoe crabs: Tachypleus tridentatus, Limulus polyphemus, Tachypleus gigas, and Tachypleus rotundicauda.
[0076]Of these, a nucleic acid fragment encoding Pro-CE derived from Tachypleus tridentatus or Limulus polyphemus is preferred, with a nucleic acid fragment encoding Pro-CE derived from Tachypleus tridentatus being more preferred.
[0077]The nucleic acid fragment of the present invention may be chemically synthesized or may be produced through a genetic engineering technique. For production through a genetic engineering technique, for example, by use of an artificially prepared primer having a nucleotide sequence defined by SEQ ID NO: 5 or 6, a target DNA fragment is amplified, through PCR technique, from a cDNA library prepared through a customary method from hemocytes (amebocytes) of a horseshoe crab such as Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas, or Tachypleus rotundicauda. The resultant PCR product may be readily isolated through molecular-weight-based separation means (e.g., gel electrophoresis).
[0078]In the present invention, the "nucleic acid fragment encoding limulus-derived Pro-CE" is more preferably any of the following nucleic acid fragments (A) to (C):
[0079](A) a DNA fragment encoding a protein having the amino acid sequence defined by SEQ ID NO: 4,
[0080](B) a DNA fragment encoding a "protein having an amino acid sequence defined by SEQ ID NO: 4 in which one or several amino acid residues are deleted, substituted, inserted, or translocated, and having activity of a limulus-derived Pro-CE,"and
[0081](C) an RNA fragment obtained through transcription of the aforementioned DNA fragment (A) or (B).
[0082]As used herein, the "DNA fragment encoding a protein having an amino acid sequence defined by SEQ ID NO: 4" is a DNA fragment encoding a Pro-CE derived from Tachypleus tridentatus.
[0083]A naturally occurring protein may include a mutation in the amino acid sequence (e.g., substitution, deletion, insertion, or translocation of amino acid residues) caused by the polymorphism or mutation of the DNA fragment encoding the protein. Meanwhile, a produced protein may include a post-translational modification (e.g., phosphorylation, glycosylation, or lipidation of amino acid residues, or hydroxylation of proline) caused by intracellular modification during purification. Although having such a mutation, some proteins are known to exhibit physiological and biological activities substantially the same as those of the protein having none of the aforementioned mutations. Thus, the "protein encoded by the DNA fragment (B)," which slightly differs from the "protein encoded by the DNA fragment (A)" in structure but has no great difference in function, can be regarded as substantially equivalent to the "protein encoded by the DNA fragment (A)." A similar logic is also applied to the case where the aforementioned mutations are intentionally introduced into an amino acid sequence of protein. In this case, a wider range of variants can be prepared. For example, as has been known, a polypeptide engineered from human interleukin 2 (IL-2) so that a certain cysteine residue in the amino acid sequence of IL-2 is substituted by serine maintains human interleukin 2 activity (Science, 224, 1431 (1984)). Such a "mutated protein" may be prepared through a known technique; for example, "site-specific mutagenesis." Also, a certain protein is known to have a peptide region that is not essential in terms of activity. Examples of such a protein include a signal peptide present in an extracellularly secreted protein, and a pro-sequence observed in a protease precursor or a similar substance. Most of these peptide regions are removed after translation or during conversion to the corresponding activated proteins. Although such proteins have a primary structure different from that of the "protein encoded by the DNA fragment (A)," the proteins have a function substantially the same as that of the "protein encoded by the DNA fragment (A)." Therefore, the "protein encoded by the DNA fragment (B)" represents these proteins.
[0084]As used herein, the term "several amino acid residues" refers to amino acid residues which are allowed to have mutations without impairing the protein activity. For example, when a protein includes 600 amino acid residues, the number of such amino acid residues is about 2 to about 30, preferably 2 to 15, more preferably 2 to 8.
[0085]The protein encoded by the DNA fragment (B) has an activity of limulus-derived Pro-CE. Activity of Pro-CE may be determined by causing Pro-CE to be co-present with a synthetic substrate (e.g., t-butoxycarbonyl-leucyl-glycyl-arginine-pNA (Boc-Leu-Gly-Arg-pNA)), Et, factor C, and factor B, and examining the reactivity of Pro-CE. Specifically, when Pro-CE has activity, pNA is released by causing Pro-CE to be co-present with the synthetic substrate, Et, and factor C. The amount of thus-produced pNA can be determined through measurement of absorbance. A specific determination method will be described in Example 2.
[0086]In the present invention, no particular limitation is imposed on the state of "co-presence," so long as co-present elements are freely allowed to be in contact with one another. Specifically, the state of "co-presence" refers to the state where Pro-CE, a synthetic substrate, Et, factor C, and factor B are freely allowed to be in contact with one another, or the state where Pro-CE, a synthetic substrate, BG, and factor G are freely allowed to be in contact with one another.
[0087]As used herein, the term "reaction" refers to a reaction in which the Pro-CE is converted into CE by causing Pro-CE to be co-present with the synthetic substrate, and CE acts on the synthetic substrate, to thereby hydrolyze the amino bonds thereof, whereby pNA is released.
[0088]The DNA fragment (A) encoding a "protein having an amino acid sequence defined by SEQ ID NO: 4" may be, for example, a DNA fragment having a nucleotide sequence of nucleotides 1 to 1,143 in SEQ ID NO: 3. Alternatively, a DNA fragment deposited in GenBank with an accession No. D161657 may also be employed.
[0089]The DNA fragment (B) encoding a "protein having an amino acid sequence defined by SEQ ID NO: 4 in which one or several amino acid residues are deleted, substituted, inserted, or translocated, and having activity of limulus-derived Pro-CE" may be, for example, the aforementioned DNA fragment (A), a DNA fragment complementary thereto, or a DNA fragment which hybridizes with any of these DNA fragments under stringent conditions.
[0090]As used herein, the term "stringent conditions" refers to conditions which allow formation of a so-called specific hybrid but do not allow formation of a non-specific hybrid (see, for example, Sambrook, J., et al., Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)). Specific examples of the "stringent conditions" include performing hybridization at 42° C. in a solution containing 50% formamide, 4×SSC, 50 mM HEPES (pH 7.0), 10×Denhardt's solution, and 100 μg/mL salmon sperm DNA, and washing at room temperature with 2×SSC and a 0.1% SDS solution and at 50° C. with 0.1×SSC and a 0.1% SDS solution.
[0091]Preferably, the nucleic acid fragment of the present invention is further linked to a DNA fragment encoding, for example, a marker peptide. Examples of the marker peptide include protein A, an insulin signal sequence, His-tag, FLAG, CBP (calmodulin-binding protein), and GST (glutathione S-transferase). The nucleic acid fragment of the present invention also encompasses an RNA fragment obtained through transcription of the aforementioned DNA fragment (A) or (B).
[0092]The nucleic acid fragment of the present invention may be employed for production of "the virus of the present invention" described hereinbelow, and thus for production of, for example, the cell of the present invention.
<2> The Virus of the Present Invention
[0093]The virus of the present invention harbors the nucleic acid fragment of the present invention.
[0094]The nucleic acid fragment of the present invention is the same as mentioned above.
[0095]The state "harboring a nucleic acid fragment" in the virus of the present invention does not exclude the state in which the virus harbors other nucleotides or nucleic acid fragments, so long as the relevant nucleic acid fragment is harbored. Thus, in addition to the relevant nucleic acid fragment, for example, other nucleic acid fragments encoding a marker peptide or a similar peptide may be harbored.
[0096]For example, the virus of the present invention also encompasses a vector harboring a linked DNA fragment formed of "the aforementioned DNA fragment (A) or (B) (i.e., the nucleic acid fragment of the present invention)" and a "DNA fragment encoding, for example, a marker peptide." When the nucleic acid fragment harbored is designed in the above manner, a protein fused with, for example, a marker peptide may be expressed. The thus-expressed protein is advantageous in that the purification, detection, analysis, etc. thereof can be facilitated. Examples of the marker peptide include protein A, an insulin signal sequence, His-tag, FLAG, CBP (calmodulin-binding protein), and GST (glutathione S-transferase). For example, a protein fused with protein A may be purified in a simple manner through affinity chromatography employing an IgG-bound solid phase. Similarly, a His-tag-fused protein may be purified with a magnetic nickel-bound solid phase, whereas a FLAG-fused protein may be purified with an anti-FLAG antibody-bound solid phase. Since a protein fused with an insulin signal sequence is extracellularly secreted (e.g., secreted in a culture medium), an extraction process including crushing of cells may be eliminated.
[0097]No particular limitation is imposed on the production method for the virus of the present invention. One exemplary method for producing the virus of the present invention will be described as follows. More specific procedure thereof will be described in the Examples.
[0098]Firstly, a nucleic acid fragment encoding limulus-derived Pro-CE is provided. In the case where the aforementioned DNA fragment (A) is employed as the nucleic acid fragment, a DNA fragment encoding a "protein having an amino acid sequence defined by SEQ ID NO: 4" is provided. In the case where the aforementioned DNA fragment (B) is employed as the nucleic acid fragment, there is provided a DNA fragment encoding a "protein having an amino acid sequence defined by SEQ ID NO: 4 in which one or several amino acid residues are deleted, substituted, inserted, or translocate (transposed), and having an activity of limulus-derived Pro-CE." No particular limitation is imposed on the type of the DNA fragment employed, so long as the DNA fragment encodes the corresponding protein. The DNA fragment includes those having a variety of nucleotide sequences attributed to degeneration of genetic codes. However, any of these DNA fragments having a specific nucleotide sequence may be employed.
[0099]The virus of the present invention can be produced through introduction of such a nucleic acid fragment into a virus.
[0100]No particular limitation is imposed on the virus into which such a nucleic acid fragment is introduced, so long as the virus can be employed for gene transfection. Particularly, a baculovirus (in particular, an NPV) is preferably employed. No particular limitation is imposed on the species of the NPV employed, so long as the NPV is a virus belonging to NPVs. For example, AcNPV or Bombyx mori NPV (BmNPV) may be employed. of these, AcNPV is preferred.
[0101]Introduction of a nucleic acid fragment into a virus may be carried out through homologous recombination by use of a transfer vector. No particular limitation is imposed on the type of the transfer vector employed. For example, pPSC8 (Protein Science), pFastBac (Invitrogen), or pVL1393 (Pharmingen) may be employed. Of these, pPSC8 is preferred. These transfer vectors may be commercially available ones.
[0102]No particular limitation is imposed on the method of homologous recombination by use of a transfer vector. A specific example thereof will be described in the Examples.
[0103]Whether or not the produced virus harbors the aforementioned DNA fragment (A) or (B) may be determined by, for example, any of the following procedures: checking that the produced virus harbors a DNA fragment encoding a limulus-derived Pro-CE through analysis of the nucleotide sequence of the virus; checking that a protein expressed by the produced virus has the amino acid sequence of limulus-derived Pro-CE; and checking that a protein expressed by the produced virus has a Pro-CE activity.
[0104]The virus of the present invention may be employed for production of "the cell of the present invention" described below, and thus employed, for example, in the method of the present invention.
<3> The Cell of the Present Invention
[0105]The cell of the present invention harbors the virus of the present invention.
[0106]"The virus of the present invention" is the same as mentioned above.
[0107]No particular limitation is imposed on the "cell" employed in the present invention, so long as the cell allows infection with the virus of the present invention, and can express the nucleic acid fragment encoding a limulus-derived Pro-CE that is harbored by the virus of the present invention. Examples of the cell include insect-derived cells. Specific examples of the insect-derived cells include an Sf9 cell.
[0108]No particular limitation is imposed on the method for causing the cell to harbor the virus of the present invention. For example, when the virus of the present invention is a NPV, the cell can be infected with the virus only by bringing the cell into contact with the virus, whereby the cell can harbor the virus. A specific method therefor will be described in the Examples hereinbelow.
[0109]Since the cell of the present invention can produce a limulus-derived Pro-CE, the cell of the present invention may be selected on the basis of the production performance as an index.
[0110]The cell of the present invention may be employed in, for example, the below-described production method of the present invention.
<4> The Production Method of the Present Invention
[0111]The production method of the present invention for producing limulus-derived Pro-CE includes at least the steps of growing the cell of the present invention, and preparing limulus-derived Pro-CE from the growth product.
[0112]"The cell of the present invention" is the same as mentioned above.
[0113]As used herein, the term "grow" refers to a concept including proliferation of cells which are transformants and growing an organism (e.g., animal or insect) into which transformant cells have been incorporated. As used herein, the term "growth product" is a concept including, for example, a culture medium (supernatant of the culture liquid) after completion of growth of transformants, cultured cells themselves, and matter secreted or discharged from an organism (e.g., animal or insect) into which the cells have been incorporated.
[0114]No particular limitation is imposed on the growth conditions (e.g., culture medium and culture conditions), so long as the cell of the present invention can grow and produce limulus-derived Pro-CE. The growth conditions may be appropriately determined in consideration of, for example, the type of a vector or cell employed. For example, culturing may be carried out at a temperature of about 20 to about 40° C.
[0115]The growth period of the cell of the present invention may also be appropriately regulated in consideration of, for example, the amount of the cell of the present invention employed, the amount of a desired Pro-CE produced, or other growth conditions.
[0116]Those skilled in the art may appropriately select the method for preparing a limulus-derived Pro-CE from the growth product from generally employed methods in consideration of the type of the growth product.
[0117]For example, in the case where Pro-CE is produced in a soluble form secreted into a culture medium (culture supernatant), the culture medium is collected and may be employed as is. In the case where Pro-CE is produced in a soluble form secreted in cytoplasm, or produced in an insoluble (membrane-bound) form, the Pro-CE may be extracted through, for example, any of the following treatments: extraction with cell crushing such as a method employing a nitrogen cavitation apparatus, homogenizing, glass beads milling, sonication, the hypotonic extraction, or freeze-thawing; extraction with a surfactant; or a combination thereof. The resultant extract may be employed, as is, as the Pro-CE.
[0118]The production method of the present invention may further include other steps, so long as the method includes at least a "step of growing the cell of the present invention, and preparing limulus-derived Pro-CE from the growth product." For example, the method may further include the step of purifying the thus-prepared Pro-CE. The purification may be incomplete (partial) purification or complete purification, and may be appropriately selected in consideration of, for example, the use purpose of the Pro-CE.
[0119]Specific examples of the purification method include salting out by the mediation of a salt such as ammonium sulfate or sodium sulfate, centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion-exchange chromatography, hydrophobic chromatography, reversed-phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, and combinations thereof.
[0120]Whether or not, for example, the thus-produced protein is formed of Pro-CE, or the protein maintains an activity of limulus-derived Pro-CE may be determined through analysis of the collected protein, in terms of, for example, amino acid sequence, molecular weight, electrophoresis features, or Western blotting employing an antibody reacting specifically to the Pro-CE.
[0121]The method of the present invention realizes very effective production of a protein which maintains Pro-CE activity.
<5> The Enzyme of the Present Invention
[0122]The enzyme of the present invention is Pro-CE produced through the production method of the present invention.
[0123]"The production method of the present invention" is the same as mentioned above.
[0124]The enzyme of the present invention may be employed in, for example, the method 1 of the present invention described below.
<6> The Method 1 of the Present Invention
[0125]The method 1 of the present invention is a method for sensitively detecting Et. A characteristic feature of the method 1 of the present invention resides in that the enzyme of the present invention is caused to be co-present with a "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with Et" in an Et-detection sample, and the Et present in the sample is detected by employing, as an index, enzymatic activity in conversion of the enzyme of the present invention to CE.
[0126]"The enzyme of the present invention" is the same as mentioned above.
[0127]In the present invention, no particular limitation is imposed on the state of "co-presence," so long as co-present elements are allowed to be in contact with one another.
[0128]For example, no particular limitation is imposed on the state of "co-presence," so long as the enzyme of the present invention, an Et-detection sample, and a "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with Et" are allowed to be in contact with one another. These elements may be caused to be co-present with one another in a solution. Alternatively, Pro-CE or a "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with Et" may be immobilized on a solid phase, and thus-immobilized Pro-CE or serine protease precursor may be respectively brought into contact with a "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with Et" or Pro-CE.
[0129]In the method 1 of the present invention, preferably, the "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with Et" is a "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with activated factor C", and "limulus-derived factor C and/or recombinant factor C."
[0130]The "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with activated factor C" is preferably limulus-derived factor B and/or recombinant factor B.
[0131]No particular limitation is imposed on the "factor C" or "factor B" employed in the method 1 of the present invention, so long as the factor maintains its function. For example, the factor C or B employed may be naturally occurring factor C or B fraction prepared through purification (e.g., chromatography) of amebocyte lysate derived from any of the four horseshoe crabs: Tachypleus tridentatus, Limulus polyphemus, Tachypleus gigas, and Tachypleus rotundicauda. Alternatively, the factor C or B employed may be recombinant factor C or B. The naturally occurring factor C or B fraction may be prepared through treatment of the aforementioned lysate with, for example, a carrier to which dextran sulfate, a sulfopropyl group, or the like is bound, or a specific adsorption carrier. The recombinant factor C may be appropriately prepared, for example, on the basis of the known amino acid sequence of naturally occurring factor C derived from Tachypleus tridentatus or Tachypleus rotundicauda. No particular limitation is imposed on the method for preparing the recombinant factor C. For example, the recombinant factor C may be prepared through the following procedure: a target nucleotide sequence for factor C having a His-tag at the C terminus is synthesized and introduced into a transfer vector (e.g., pPSC8, product of Takara Bio Inc.); Sf9 cells are co-transfected with the resultant expression vector (Factor C/pPSC8) DNA fragment and a baculovirus (AcNPV) DNA fragment; and the virus fluid obtained from the resultant culture supernatant is purified, followed by amplification. The recombinant factor B may also be prepared in a manner similar to that described above. The amino acid sequence of factor C or factor B and the gene coding therefor have already been known. Factor C is commercially available, and the commercial product will be described in the Examples hereinbelow. The nucleotide sequence of the gene for factor B is shown in SEQ ID NO: 15, and the amino acid sequence of factor B is shown in SEQ ID NO: 16. The recombinant of such a factor may be produced on the basis of the corresponding sequence data through a process substantially the same as that for the aforementioned enzyme of the present invention. It will be readily appreciated by those skilled in the art that a protein produced on the basis of a nucleotide sequence which, as a result of degeneration of genetic codes, differs from the known nucleotide sequence of factor C or the nucleotide sequence defined by SEQ ID NO: 15 may be employed in the present invention as factor C or B, so long as the protein has an effect intrinsic to factor C or B.
[0132]Operation of the reconstruction system does not require provision of insect cells as a reaction site. For facilitation of at least triggering of cascade reaction, sequential activation of serine protease precursors, and reaction of CE in a cell-free system, preferably, conditions for the operation include, for example, constant heating and co-presence of ions of a metal such as an alkaline earth metal (e.g., calcium, strontium, barium, beryllium, or magnesium) or an alkali metal (e.g., lithium, sodium, or potassium).
[0133]In the method 1 of the present invention, more preferably, the enzyme of the present invention, recombinant factor C, and recombinant factor B are exclusively caused to be co-present as cascade reaction proteins.
[0134]As used herein, the term "cascade reaction" refers to the following reaction "1." and/or reaction "2.":
[0135]1. a series of reactions in which factor C (Et-sensitive factor, molecular weight: 123,000) present in amebocyte lysate is activated through addition of Et to the lysate, to thereby form activated factor C; the activated factor C hydrolyzes a specific site of factor B (molecular weight: 64,000) to thereby form activated factor B; the activated factor B activates Pro-CE (molecular weight: 54,000) to thereby convert it into CE; and the CE hydrolyzes specific sites in a loop cross-linked by disulfide bonds of coagulogen (coagulated protein, molecular weight: 19,723); i.e., hydrolyzes the bond between Arg18 and Thr19 and the bond between Arg46 and Gly47, to thereby release peptide C (28 amino acid residues) represented by H-Thr19 . . . Arg46-OH and to convert the remaining portion into coagulin gel; and
[0136]2. a series of reactions in which factor G (BG-sensitive factor) present in amebocyte lysate is activated through addition of BG to the lysate; the activated factor G activates Pro-CE to thereby convert it into CE; and the CE hydrolyzes specific sites in a loop cross-linked by disulfide bonds of coagulogen, to thereby form coagulin gel.
[0137]The term "cascade reaction protein" refers to a protein involved in "cascade reaction"; i.e., a serine protease precursor (factor C, factor B, factor G, or Pro-CE). Specifically, the cascade reaction proteins in the aforementioned cascade reaction "1." are factor C, factor B, and Pro-CE, and the cascade reaction proteins in the cascade reaction "2." are factor G and Pro-CE.
[0138]As shown in the Examples hereinbelow, the origins of the genetically engineered coagulation factors employed in the method 1 of the present invention may be different from one another. For example, the Et detection method may include the step of causing the enzyme of the present invention to be co-present with factor C derived from Tachypleus rotundicauda and factor B derived from Limulus polyphemus.
[0139]The method 1 of the present invention can be readily carried out by means of the below-described kit 1 of the present invention.
<7> The Kit 1 of the Present Invention
[0140]The kit 1 of the present invention is an Et detection kit for carrying out the method 1 of the present invention. A characteristic feature of the kit 1 of the present invention resides in that the kit includes at least the enzyme of the present invention and a "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with Et."
[0141]"The enzyme of the present invention" is the same as mentioned above.
[0142]The "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with activated factor C" is preferably limulus-derived factor B and/or recombinant factor B.
[0143]Preferably, the kit 1 of the present invention includes, as cascade reaction proteins, exclusively the enzyme of the present invention, recombinant factor C, and recombinant factor B.
[0144]Those skilled in the art may appropriately employ the kit 1 of the present invention on the basis of the method 1 of the present invention.
[0145]The terms used in the kit 1 of the present invention such as "factor C," "factor B," and "cascade reaction" have the same meanings as defined above in the method 1 of the present invention. As described above, reagents, etc. employed for carrying out the method 1 of the present invention (e.g., the aforementioned synthetic chromogenic substrate (X-A-Y), buffer, diluent, salt, and limulus-derived amebocyte lysate) may be included in the kit 1 of the present invention, which may be selected in consideration of the mode of the method 1 of the present invention performed by means of the kit.
<8> The Method 2 of the Present Invention
[0146]The method 2 of the present invention is a method for detecting BG. A characteristic feature of the method 2 of the present invention resides in that the enzyme of the present invention is caused to be co-present with a "serine protease precursor which expresses an activity of converting pro-clotting enzyme to clotting enzyme upon contact with BG" in a BG-detection sample, and BG present in the sample is detected by employing, as an index, enzymatic activity induced through conversion of the enzyme of the present invention to clotting enzyme.
[0147]"The enzyme of the present invention" is the same as mentioned above.
[0148]In the method 2 of the present invention, preferably, the "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with BG" is limulus-derived factor G and/or recombinant factor G.
[0149]No particular limitation is imposed on the "factor G" employed in the method 2 of the present invention, so long as the factor maintains its function. For example, the factor G employed may be a naturally occurring factor G fraction prepared through purification (e.g., chromatography) of amebocyte lysate derived from any of the aforementioned four horseshoe crabs. Alternatively, the factor G employed may be recombinant factor G. Recombinant factor G is a protein formed of subunits α and β, and the respective subunits may be produced through the following procedures.
[0150]Firstly, a DNA fragment encoding subunit α of limulus-derived factor G is provided. The DNA fragment may be, for example, a DNA fragment deposited in GenBank with an accession No. 16622 (SEQ ID NO: 17, the amino acid sequence corresponding to the DNA fragment is shown in SEQ ID NO: 18). The DNA fragment is treated with BamHI/Hind III, and DNA fragments having a target gene sequence are collected. The sample is blunt-ended and then ligated through mixing with Nru I-treated pPSC8 (transfer vector). Subsequently, E. coli JM109 is transformed with the ligation product, to thereby yield a transformant. Plasmid in which fragments having a target size have been determined is purified. Sf9 cells are co-transfected with the thus-selected expression vector (Factor G-α/pPSC8) DNA fragment and a baculovirus (AcNPV) DNA fragment. Thereafter, the virus fluid obtained from the resultant culture supernatant is purified, followed by amplification. Then, expres SF+ cells are infected with the virus fluid, and the resultant culture liquid is centrifuged, to thereby yield a supernatant fraction and a precipitate fraction. Subunit α of factor G may be prepared from these fractions. Subunit β may be prepared by performing the same procedure as for subunit α, except that the subunit-α-encoding DNA fragment is replaced with a DNA fragment encoding subunit β of limulus-derived factor G. The subunit-β-encoding DNA fragment may be, for example, a nucleotide sequence deposited in GenBank with an accession No. 16623 (SEQ ID NO: 19) (the amino acid sequence corresponding thereto is shown in SEQ ID NO: 20). It will be readily appreciated by those skilled in the art that a protein produced on the basis of nucleotide sequences which, as a result of degeneration of genetic codes, differ from the nucleotide sequences of SEQ ID NOs: 16 and 17 may be employed in the present invention as factor G, so long as the protein has an effect intrinsic to factor G.
[0151]Operation of the reconstruction system does not require provision of a certain type of cells (e.g., insect cells) as a reaction site. For facilitation of at least triggering of cascade reaction, sequential activation of serine protease precursors, and reaction of CE in a cell-free system, preferably, conditions for the operation include, for example, constant heating and co-presence of ions of a metal such as an alkaline earth metal or an alkali metal.
[0152]In the method 2 of the present invention, more preferably, the enzyme of the present invention and recombinant factor G are exclusively caused to be co-present as cascade reaction proteins. Similar to the case of the method 1 of the present invention, the enzyme of the present invention and recombinant factor G may be derived from the same origin or different origins.
[0153]The terms used in the method 2 of the present invention such as "co-present," "cascade reaction," and "cascade reaction protein" have the same meanings as defined above in the method 1 of the present invention.
[0154]The method 2 of the present invention may be employed in the below-described kit 2 of the present invention.
<9> The Kit 2 of the Present Invention
[0155]The kit 2 of the present invention is a BG detection kit for carrying out the method 2 of the present invention. A characteristic feature of the kit 2 of the present invention resides in that the kit includes at least the enzyme of the present invention and a "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with BG."
[0156]"The enzyme of the present invention" is the same as mentioned above.
[0157]In the kit 2 of the present invention, preferably, the "serine protease precursor which expresses an activity of converting Pro-CE to CE upon contact with BG" is limulus-derived factor G and/or recombinant factor G.
[0158]More preferably, factor G employed in the kit is in a recombinant form. In a most preferred mode of the kit 2 of the present invention, all the factors G molecules included in the kit are in a recombinant form. That is, more preferably, the kit 2 of the present invention includes, as cascade reaction proteins, exclusively the enzyme of the present invention and recombinant factor G.
[0159]Those skilled in the art may appropriately employ the kit 2 of the present invention on the basis of the method 2 of the present invention.
[0160]The term "factor G" used in the kit 2 of the present invention has the same meaning as described in the method 2 of the present invention. The terms "cascade reaction" and "cascade reaction protein" have the same meanings as defined above in the method 1 of the present invention. As described above, reagents, etc. employed for carrying out the method 2 of the present invention (e.g., the aforementioned synthetic chromogenic substrate (X-A-Y), buffer, diluent, salt, and limulus-derived amebocyte lysate) may be included in the kit 2 of the present invention, which may be selected in consideration of the mode of the method 2 of the present invention performed by means of the kit.
EXAMPLES
[0161]The present invention will next be described in more detail by way of examples.
Example 1
Expression of Pro-CE by Use of Insect Cells
[0162]A cDNA fragment represented by SEQ ID NO: 3 (having a nucleotide sequence of nucleotides 1 to 1125 in SEQ ID NO: 3, a His-Tag sequence being attached to the C-terminus) was kindly offered by Dr. Tatsushi MUTA (Department of Molecular and Cellular Biochemistry, Kyushu University Graduate School of Medical Science; currently, Department of Bio-Science, Tohoku University Graduate School). The cDNA fragment had been prepared through a method disclosed in Non-Patent Document 2. The cDNA fragment was introduced into a transfer vector (pPSC8), and a clone having a predetermined nucleotide sequence was selected. The thus-selected expression vector (PC/pPSC8) DNA fragment and a baculovirus (AcNPV) DNA fragment were co-transfected into Sf9 cells. The virus fluid obtained from the culture supernatant was purified and amplified. The viral DNA fragment was extracted from the cells infected with the baculovirus, and sequenced. Insect cells (expresSF+, registered trademark, product of protein Science) were infected with the thus-obtained virus fluid, and the expression product was analyzed through Western blotting. Details of these steps will next be described.
1. Construction of Expression Vector
[0163]PC/pUC118 (20 ng/μg) (1 μL), 2.5 mM dNTP (12 μL), KOD buffer # (15 μL), 25 mM magnesium chloride solution (2 μL), PC-F and PC-R (4 pmol/μL) (each 2.5 μL), KOD DNA polymerase (product of Toyobo) (1 μL), and sterilized pure water (24 μL) were added to a 0.2-mL sample tube under stirring, and the mixture was subjected to PCR. The presence of a target gene in the PCR product was confirmed, and the product was diluted with TE buffer to the total volume of 16 μL. Then, 100 mM ATP (1 μL), 10× Buffer (1 μL), and T4Polynucleotide Kinase (product of Takara Bio) were added to the obtained PCR product, and the mixture was incubated at 37° C. for 30 minutes. The PCR product was purified and subjected to ligation through mixing with Sma I-treated pPSC12. E. coli JM109 was transformed with the ligation product, to thereby form a transformant (PC/pPSC12). PC/pPSC12 was digested with Xba I/Bgl II, and a fragment containing the target gene was recovered. The thus-produced fragment was mixed with pPSC8 which had been treated with the same enzyme, and the resultant mixture was subjected to ligation. E. coli JM109 was transformed with the ligation product, to thereby form a transformant. Plasmids in which fragments of the target size had been confirmed were purified, and sequenced. The sequencing was performed by use of the below-described primers and ABI Prism Big Dye Terminator Cycle Sequencing Kit Ver. 3 (Applied Biosystems). The analysis was performed by means of an automated sequencer ABI Prism 310 Genetic Analyzer (Applied Biosystems). Sequences of the primers are shown in the following sequence list by SEQ ID NOs: 5 to 10.
SEQ ID NO: 5: PC-F
SEQ ID NO: 6: PC-R
SEQ ID NO: 7: PSC2
SEQ ID NO: 8: PSC4
SEQ ID NO: 9: PC 453/472-F
SEQ ID NO: 10: PC 683/664-R
[0164]A clone in which insertion of a target gene had been confirmed was inoculated to an LB medium (100 mL) containing 50 μg/mL ampicillin, and cultivated at 30° C. for one night. Proliferated cells were collected, and plasmids were purified in accordance with an instruction manual of Plasmid Midi Kit (QIAGEN).
2. Co-Transfection
[0165]To Sf9 cells (1.0×106) plated in a 25-cm2 flask was added a serum-free Sf-900 II medium (product of Invitrogen) (200 μL) containing an expression vector harboring a cDNA fragment encoding Pro-CE (2.3 μg), a linear AcNPV DNA (85 ng), and LIPOFECTIN Reagent (product of Invitrogen) (5 μL). After the culture had been allowed to stand at 28° C. for six hours, a serum-free Sf-900 II medium was further added so as to adjust the volume of the culture liquid to 5 mL. The culture was further cultivated at 28° C. for five days, and the culture supernatant was collected. The supernatant was employed as a co-transfection solution.
3. Purification of Recombinant Virus
[0166]The recombinant virus was purified through the plaque assay method. The specific procedure is as follows.
[0167]Sf9 cells (2.0×106) were plated onto a plate (diameter: 60 mm) and allowed to stand at 28° C. for one hour, whereby the cells were adhered to the bottom surface. The aforementioned co-transfection solution was diluted with a serum-free Sf-900 II medium at dilution factors of 104, 105, 106, and 107. An aliquot (1 mL) of each of these diluted solutions was added to the cells, followed by gentle shaking at room temperature for one hour. After removal of the plate supernatant (virus fluid), a serum-free Sf-900 II medium (4 mL) containing 0.5% SeaKemGTG agarose (product of BMA) was added to the plate, and stationary culture was performed at 28° C. for eight days. From each culture medium, six plaques of infected insect cells including no polyhedra were collected. The plaques of each medium were suspended in a serum-free Sf-900 II medium (1 mL), to thereby obtain a virus fluid.
4. Amplification of Recombinant Virus
[0168]Next, amplification of the recombinant virus (preparation of recombinant virus fluid) was performed. The specific procedure is as follows.
[0169]To Sf9 cells (2.0×106) plated in a 25-cm2 flask was added each (0.5 mL) of the aforementioned virus fluids, followed by stationary cultivation at 28° C. for one hour. A serum-free Sf-900 II medium was added to the culture so as to adjust the volume of the culture liquid to 5 mL, and the culture was further stationary-cultivated for three days, to thereby yield a first-generation virus fluid.
[0170]To Sf9 cells (6.0×106) plated in a 75-cm2 flask was added the entirety of the aforementioned first-generation virus fluid, followed by stationary cultivation at 28° C. for one hour. Subsequently, a serum-free Sf-900 II medium (10 mL) was added to the culture, followed by stationary cultivation for three days. After completion of cultivation, the culture supernatant was recovered and centrifuged at 3,000×g and 4° C. for 15 minutes, to thereby fractionate into the supernatant and the precipitate. The culture supernatant was recovered and employed as a second-generation virus fluid.
5. Production of Recombinant Virus Fluid
[0171]Insect cells (expresSF+) which were in the logarithmic growth phase during cultivation were diluted with a serum-free Sf-900 II medium so as to adjust the concentration to 1.5×106 cells/mL, and the diluted product (50 mL) was placed in a 125-mL Erlenmeyer flask. The aforementioned second-generation virus fluid (0.5 mL) was added thereto, and the mixture was subjected to shake cultivation at 130 rpm and 28° C. for three days. After completion of cultivation, the culture liquid was centrifuged at 10,000×g and 4° C. for 30 minutes, to thereby fractionate into the supernatant and the precipitate. The culture supernatant was recovered and employed as a third-generation virus fluid.
6. Confirmation of Gene Insertion
[0172]Subsequently, insertion of a DNA fragment into a cell was confirmed through the following procedure.
[0173]The thus-recovered third-generation virus fluid (0.7 mL) was placed in a 1.5-mL microtube, and an equiamount of 20% polyethylene glycol and 1M sodium chloride solution were added to the microtube, followed by sufficiently mixing. The mixture was allowed to stand for one hour. Thereafter, the culture liquid was centrifuged at 10,000×g for 10 minutes, to thereby fractionate into the supernatant and the precipitate. The precipitate was recovered and, in accordance with a manual of QIAamp DNA Miki Kit (QIAGEN), dissolved in Buffer ALT (180 μL), whereby a viral DNA fragment was extracted. PCR was performed in the following manner by use of the thus-extracted viral DNA fragment as a template and the following primers.
SEQ ID NO: 11: PSC N3F
SEQ ID NO: 12: PSC N3R
[0174]To a 0.2-mL sample tube, the aforementioned viral DNA fragment (1 μL), 10×PCR buffer for KOD -Plus- (5 μL), 2 mM dNTPs (5 μL), 25 mM magnesium sulfate solution (2 μL), primers PSC N3F and PSC N3R (4 pmol/mL, 3.75 μL, each), KOD -Plus-DNA polymerase (product of TOYOBO) (1 μL), and sterilized pure water (19.5 μL) were added, and the mixture was sufficiently stirred. The mixture was subjected to PCR for 30 cycles, each cycle consisting of 94° C. for one minute, 58° C. for one minute, and 72° C. for four minutes.
[0175]The PCR product (10 μL) was subjected to electrophoresis on agarose gel, and the length of the amplified fragments was determined. A PCR product of a fragment having a target length was purified, and the sequences of the N-terminus and the C-terminus were determined, through use of the same reagents and apparatuses as employed in the aforementioned "1. Construction of expression vector." The following primers represented by SEQ ID NOs: 13 and 14 were employed.
SEQ ID NO: 13: PSC NF2
SEQ ID NO: 14: PSC 3
7. Titer Determination
[0176]Sf9 cells (2.0×106) were plated onto a plate (diameter: 60 mm) and allowed to stand at 28° C. for one hour, whereby the cells were adhered to the bottom surface. Subsequently, the culture liquid was removed. Separately, the third-generation virus fluid was diluted with a serum-free Sf-900 II medium at dilution factors of 105, 106, 107, and 108. An aliquot (1 mL) of each of these solutions was added to the plate, followed by gentle shaking at room temperature for one hour. After removal of the plate supernatant (virus fluid), a serum-free Sf-900 II medium (4 mL) containing 0.5% SeaKemGTG agarose (product of BMA) was added to the plate, and stationary culture was performed at 28° C. for seven days. In each culture medium, the number of observed plaques was counted, thereby determining the titer.
8. Expression Test
[0177]Insect cells (expresSF+) were diluted with a serum-free Sf-900 II medium so as to adjust the concentration to 1.5×106 cells/mL, and the diluted product (100 mL/per flask) was placed in nine 250-mL Erlenmeyer flasks. The aforementioned third-generation virus fluid was added thereto so as to attain MOIs of 0.2, 1, and 5 (3 flasks each), respectively. Each mixture was subjected to shake cultivation at 130 rpm and 28° C. for 48, 72, and 96 hours. After completion of cultivation, each culture liquid was centrifuged at 3,000×g and 4° C. for 20 minutes, to thereby fractionate into the supernatant and the precipitate.
9. Detection of Expression Product
[0178]Each of the samples collected in "8. Expression test" above was subjected to SDS-PAGE through a routine method. A protein was transferred to a blotting membrane through the semi-dry blotting method, and western blotting was performed under the following conditions. Note that the "DNA fragment encoding Pro-CE" incorporated into the virus was designed so as to express a His-Tag-bound protein.
Sample Treatment: the Supernatant was Mixed with Laemmli Sample Buffer (product of BIO-RAD), and the mixture was heated at 99° C. for three minutes. The precipitate (200 μL) was mixed with PBS (400 μL), to thereby form an aqueous suspension. Laemmli Sample Buffer was added to the suspension, and the mixture was heated at 99° C. for three minutes.Amount of applied sample: 20 μL/laneSDS-PAGE gel concentration: 10 to 20% gel (product of BIO-RAD)Voltage application in SDS-PAGE: 150V, CVBlotting membrane: PVDFVoltage application in blotting: 15V, CV, 30 minutesPrimary antibody: Penta•His Antibody (product of QIAGEN)Secondary antibody: Goat Anti-Mouse IgG(H+L)-HRP Conjugate (product of BIO-RAD)Detection: Immobilon Western Chemiluminescent HRP Substrate (product of Milipore)
10. Results
[0179]FIG. 1 shows the results of sequence analysis of the target sequence of the recombinant virus. The upper column shows the analysis results, and the lower columns show the sequence of Pro-CE. As is clear from FIG. 1, the recombinant virus was found to have the same N-terminal sequence and C-terminal sequence as those of Pro-CE. Thus, the presence of a DNA fragment having a nucleotide sequence encoding Pro-CE was confirmed in the recombinant virus.
[0180]The titer was 0.7×108 pfu/mL.
[0181]In the results of "9. Detection of expressed product" above, a band attributed to reaction with an anti-His-Tag antibody was observed at a target position (about 40 kDa) (FIG. 2). In FIG. 2, M represents a molecular weight marker, 48, 72, and 96 represent 48-hour-infection-lane, 72-hour-infection-lane, and 96-hour-infection-lane, respectively, S represents a non-virus-infected lane, and A represents a wild-type-virus-infected lane. As is clear from FIG. 2, expression of Pro-CE was verified.
Example 2
Detection of Recombinant Pro-CE Activity
[0182]Insect cells (expresSF+) were diluted with a serum-free Sf-900 II medium so as to adjust the concentration to 1.5×106 cells/mL, and the diluted product (100 mL/per flask) was placed in nine 250-mL Erlenmeyer flasks. The aforementioned third-generation virus fluid was added thereto so as to attain MOIs of 0.2, 1, and 5 (3 flasks each), respectively. Each mixture was subjected to shake cultivation at 130 rpm and 28° C. for 48, 72, and 96 hours. After completion of cultivation, each culture liquid was centrifuged at 3,000×g and 4° C. for 20 minutes, to thereby fractionate into the supernatant and the precipitate. The supernatant was preserved in a frozen state.
Sample 1: MOI=0.2, 48 hoursSample 2: MOI=0.2, 72 hoursSample 3: MOI=0.2, 96 hoursSample 4: MOI=1, 48 hoursSample 5: MOI=1, 72 hoursSample 6: MOI=1, 96 hoursSample 7: MOI=5, 48 hoursSample 8: MOI=5, 72 hoursSample 9: MOI=5, 96 hoursSample 10: non-virus-infected cellsSample 11: wild-type-virus-infected cells
Reagents
[0183]DS-3GII fraction: Factor G derived from a horseshoe crab hemocyte extract [product fractionated/purified with dextran sulfate Sepharose CL-6B (hereinafter referred to as DS-Sepharose)]DS-10AII fraction: Pro-CE derived from a horseshoe crab hemocyte extract (product purified with DS-Sepharose)BG: CSBG (1,495 ng/vial) dissolved in distilled water (1.495 mL), followed by ×10-stepwise dilution1. Reactivity of Supernatant Fractions Employing Factor G at a BG Concentration of 1 to 100 ng/mL
[0184]Firstly, there was investigated whether or not the expressed protein maintained Pro-CE activity in each of the supernatants of the aforementioned nine samples.
[0185]Each supernatant fraction recovered after culture was 10-fold diluted with 50 mL Tris-HCl buffer (pH: 7.5) containing 150 mM NaCl. To each diluted product (25 μL), there were added DS-3GII fraction (25 μL), dextran (final concentration: 2.4%), Tris-HCl buffer (pH: 8.0) (final concentration: 80 mM), MgSO4 (final concentration: 64 mM), CaCl2 (final concentration: 0.4 mM), Na2SO4 (final concentration: 8 mM), distilled water for injection (15 μL), Boc-Leu-Gly-Arg-pNA substrate (see the aforementioned Patent Document 1) (final concentration: 0.24 mM), and a BG solution (0, 1, 10, or 100 ng/mL) (25 μL). The total volume of the sample was adjusted to 125 μL, and the sample was transferred to a Wellreader SK603, where it was allowed to react at 37° C. for two hours. The absorbance of the sample was automatically determined at a measurement wavelength of 405 nm (control wavelength: 492 nm). As a positive control, a DS-10AII fraction was employed. The measurement was carried out twice, and the average absorbance was calculated. The results are shown in FIGS. 3 and 4 and Table 1.
TABLE-US-00001 TABLE 1 [Endpoint Assay] Reactivity (A 405 to Sample Sample No. 492 nm) Average CSBG Recombinant 1) MOI = 0.2, 48 h 0.029 0.029 0.029 (0 ng/ Pro-CE 2) MOI = 0.2, 72 h 0.029 0.030 0.030 mL) (×10 diluted) 3) MOI = 0.2, 96 h 0.031 0.031 0.031 4) MOI = 1, 48 h 0.030 0.032 0.031 5) MOI = 1, 72 h 0.030 (0.041) 0.030 6) MOI = 1, 96 h 0.031 0.030 0.031 7) MOI = 5, 48 h 0.030 0.030 0.030 8) MOI = 5, 72 h 0.030 0.030 0.030 9) MOI = 5, 96 h 0.031 0.031 0.031 (×10 diluted) 10) Non-infected 0.029 0.030 0.030 cells 11) Wild-type- 0.030 0.029 0.030 infected DS-10AII fraction (control) 0.040 0.041 0.041 CSBG Recombinant 1) MOI = 0.2, 48 h 0.308 0.317 0.313 (1 ng/ Pro-CE 2) MOI = 0.2, 72 h 0.462 0.461 0.462 mL) (×10 diluted) 3) MOI = 0.2, 96 h 0.396 0.397 0.397 4) MOI = 1, 48 h 0.332 0.331 0.332 5) MOI = 1, 72 h 0.382 0.380 0.381 6) MOI = 1, 96 h 0.376 0.370 0.373 7) MOI = 5, 48 h 0.359 0.365 0.362 8) MOI = 5, 72 h 0.401 0.396 0.399 9) MOI = 5, 96 h 0.344 0.344 0.344 (×10 diluted) 10) Non-infected 0.052 0.053 0.053 cells 11) Wild-type- 0.051 0.052 0.052 infected DS-10AII fraction (control) 0.510 0.481 0.496 CSBG Recombinant 1) MOI = 0.2, 48 h 0.567 0.578 0.573 (10 ng/ Pro-CE 2) MOI = 0.2, 72 h 0.612 0.642 0.627 mL) (×10 diluted) 3) MOI = 0.2, 96 h 0.606 0.628 0.617 4) MOI = 1, 48 h 0.516 0.518 0.517 5) MOI = 1, 72 h 0.589 0.588 0.589 6) MOI = 1, 96 h 0.595 0.589 0.592 7) MOI = 5, 48 h 0.527 0.555 0.541 8) MOI = 5, 72 h 0.605 0.597 0.601 9) MOI = 5, 96 h 0.552 0.558 0.555 (×10 diluted) 10) Non-infected 0.084 0.084 0.084 cells 11) Wild-type- 0.083 0.083 0.083 infected DS-10AII fraction (control) 0.699 0.725 0.712
[0186]As shown in Table 1, the virus-infected cell culture supernatants of samples 1 to 9 each contain the enzyme in almost the same amount, indicating the presence of expression of Pro-CE in all fractions. In particular, sample 2 exhibited strong activity.
[0187]Table 2 and FIG. 5 show the results of comparison of recombinant Pro-CE of sample 2 with DS-10AII fraction in terms of in enzymatic activity.
TABLE-US-00002 TABLE 2 [Endpoint assay] CSBG Reactivity concentration (A 405 to Sample (ng/mL) 492 nm) Average ΔA DS- DS-10AII 0 0.056 0.060 0.058 -- 3GII (X1) (X1) 1 0.440 0.434 0.437 0.379 10 0.969 1.002 0.986 0.928 Recombinant 0 0.051 0.049 0.050 -- ProCE (X5) 1 0.480 0.487 0.484 0.434 10 0.988 0.986 0.987 0.937
[0188]As is clear from Table 2, the recombinant Pro-CE exhibited an enzymatic activity almost equivalent to that of Pro-CE derived from a horseshoe crab hemocyte extract.
2. Reactivity of Supernatant Fractions Employing Factors B and C at an Et Concentration of 1 to 100 ng/mL
[0189]The recombinant Pro-CE or the DS10-AII fraction (positive control, Pro-CE-eluted fraction); a DS-12BCI fraction (factor-B-C-eluted fraction) (25 μL) instead of factors B and C; and Et were added to the diluted products obtained in [1.] above. The Et concentration was adjusted to 0, 1, 10, or 100 ng/mL. The other mixture components were added in amounts and at concentrations equivalent to those employed in [1.]. The total volume of each sample was adjusted to 125 μL, and the sample was transferred to a Wellreader SK603, where it was allowed to react at 37° C. for 30 minutes. The absorbance of the sample was automatically determined at a measurement wavelength of 405 nm (control wavelength: 492 nm). The measurement was carried out twice, and the average absorbance was calculated. The results are shown in FIGS. 6 and 7 and Table 3.
TABLE-US-00003 TABLE 3 [Endpoint Assay] Reactivity (A 405 to Sample Sample No. 492 nm) Average Endotoxin Recombinant 1) MOI = 0.2, 48 h 0.036 0.035 0.036 (0 ng/mL) Pro-CE 2) MOI = 0.2, 72 h 0.036 0.037 0.037 (×10 diluted) 3) MOI = 0.2, 96 h 0.038 0.037 0.038 4) MOI = 1, 48 h 0.037 0.038 0.038 5) MOI = 1, 72 h 0.037 0.039 0.038 6) MOI = 1, 96 h 0.037 0.037 0.037 7) MOI = 5, 48 h 0.038 0.038 0.038 8) MOI = 5, 72 h 0.038 0.038 0.038 9) MOI = 5, 96 h 0.038 0.039 0.039 (×10 diluted) 10) Non-infected 0.036 0.038 0.037 cells 11) Wild-type- 0.039 0.038 0.039 infected DS-10AII fraction (control) 0.044 0.044 0.044 Endotoxin Recombinant 1) MOI = 0.2, 48 h 0.066 0.067 0.067 (1 ng/mL) Pro-CE 2) MOI = 0.2, 72 h 0.084 0.083 0.084 (×10 diluted) 3) MOI = 0.2, 96 h 0.070 0.072 0.071 4) MOI = 1, 48 h 0.070 0.071 0.071 5) MOI = 1, 72 h 0.075 0.075 0.075 6) MOI = 1, 96 h 0.069 0.069 0.069 7) MOI = 5, 48 h 0.074 0.075 0.075 8) MOI = 5, 72 h 0.074 0.077 0.076 9) MOI = 5, 96 h 0.065 0.069 0.067 (×10 diluted) 10) Non-infected 0.049 0.054 0.052 cells 11) Wild-type- 0.051 0.053 0.052 infected DS-10AII fraction (control) 0.156 0.143 0.150 Endotoxin Recombinant 1) MOI = 0.2, 48 h 0.306 0.276 0.291 (10 ng/mL) Pro-CE 2) MOI = 0.2, 72 h 0.420 0.395 0.408 (×10 diluted) 3) MOI = 0.2, 96 h 0.317 0.317 0.317 4) MOI = 1, 48 h 0.306 0.302 0.304 5) MOI = 1, 72 h 0.333 0.338 0.336 6) MOI = 1, 96 h 0.295 0.297 0.296 7) MOI = 5, 48 h 0.333 0.329 0.331 8) MOI = 5, 72 h 0.339 0.337 0.338 9) MOI = 5, 96 h 0.271 0.268 0.270 (×10 diluted) 10) Non-infected 0.146 0.147 0.147 cells 11) Wild-type- 0.146 0.145 0.146 infected DS-10AII fraction (control) 0.722 0.724 0.723 Endotoxin Recombinant 1) MOI = 0.2, 48 h 0.634 0.634 0.634 (100 ng/mL) Pro-CE 2) MOI = 0.2, 72 h 0.736 0.806 0.771 (×10 diluted) 3) MOI = 0.2, 96 h 0.727 0.686 0.707 4) MOI = 1, 48 h 0.662 0.650 0.656 5) MOI = 1, 72 h 0.707 0.678 0.693 6) MOI = 1, 96 h 0.677 0.666 0.672 7) MOI = 5, 48 h 0.729 0.693 0.711 8) MOI = 5, 72 h 0.720 0.699 0.710 9) MOI = 5, 96 h 0.710 0.704 0.707 (×10 diluted) 10) Non-infected 0.306 0.301 0.304 cells 11) Wild-type- 0.300 0.302 0.301 infected DS-10AII fraction (control) 0.733 0.757 0.745
[0190]As is clear from Table 3, all virus-infected cell culture supernatants of samples each contain Pro-CE. In particular, sample 2 exhibited strong activity.
Example 3
Detection of Expression of Activity in a Complete Reconstitution System Employing Recombinant Pro-CE and Recombinant Factor G
Reagents
[0191](1) recombinant factor G (culture supernatant; α-subunit:β-subunit=2:1)(2) recombinant Pro-CE (culture supernatant of sample 2 in Example 2)(3) BG:CSBG (1,495 ng/vial) dissolved in distilled water (1.495 mL), followed by ×10-stepwise dilution
[0192]Each of the culture supernatants (1) and (2), which had been produced through the method disclosed in Japanese Patent Application Laid-Open (kokai) No. 2006-271384 (Patent Document 2), was five-fold diluted with Tris-HCl buffer (pH: 7.5) (50 mL) containing 150 mM NaCl. In this experiment, a nucleotide sequence represented by SEQ ID NO: 21 was employed as a DNA fragment encoding α-subunit of factor G. To the thus-diluted recombinant factor G (25 μL), recombinant Pro-CE (25 μL), Tris-HCl buffer (pH: 8.0) (final concentration: 80 mM), MgSO4 (final concentration: 64 mm), CaCl2 (final concentration: 0.4 mM), Na2SO4 (final concentration: 8 mm), distilled water for injection (15 μL), dextran (final concentration: 2.4%), Boc-Leu-Gly-Arg-pNA substrate (final concentration: 0.24 mm), and a BG solution (0, 1, or 10 ng/mL) (25 μL) were added. The total volume of each sample was adjusted to 125 μL, and the sample was transferred to a Wellreader SK603, where it was allowed to react through a routine procedure. The absorbance of the sample was automatically determined at a measurement wavelength of 405 nm (control wavelength: 492 nm). The measurement was carried out twice, and the average absorbance was calculated. The results are shown in Table 4 and FIG. 8.
TABLE-US-00004 TABLE 4 [Endpoint assay] CSBG Reactivity concentration (A 405 to 492 nm) Average ΔA Sample (ng/mL) (×103) (×103) (×103) Recombinant Recombinant 0 0.079 0.076 0.078 -- Factor G ProCE (X5) 1 0.140 0.138 0.139 0.061 (×5) 10 0.543 0.520 0.532 0.454
[0193]As is clear from Table 4 and FIG. 8, similar to the case of a factor contained in amebocyte lysate (native factor LAL), reaction of BG proceeds in a concentration-dependent manner through employment of recombinant factor G and recombinant Pro-CE in combination. Also, the same enzymatic activity as that of native factor LAL was found to be expressed.
Example 4
Detection of Et Reaction by Use of a Complete Reconstitution System Employing Recombinant Pro-CE, Recombinant Factor B, and Recombinant Factor C
[0194]A nucleotide sequence targeting for expressing factor B in which a His-Tag sequence is attached to the C-terminus was synthesized, and the synthesized nucleotide sequence was introduced into a transfer vector (pPSC8). The thus-obtained expression vector (factor B/pPSC8) DNA fragment and a baculovirus (AcNPV) DNA fragment were co-transfected into Sf9 cells. The virus fluid obtained from the culture supernatant was purified and amplified. The viral DNA fragment was extracted from the cells and sequenced, to thereby determine the sequences of the N- and C-terminuses of the introduced gene fragment. The expresSF+ cells (equivalent to 100 mL of culture liquid) were caused to be infected with the thus-produced recombinant virus so as to attain MOIs of 0.02, 0.1, and 0.5, and culture supernatants and precipitates were recovered at hour 48, hour 72, and hour 96. The recovered expression products were analyzed through western blotting employing an anti-His-Tag antibody for confirming expression. Subsequently, Et (0, 0.01, 0.1, 1, 10, or 100 μg/mL) was added to a reaction system in the presence of this factor, recombinant factor C (product of PyroGene), and recombinant ProCE, and the system was allowed to react at 37° C. for one hour. Through analyzing the ability to hydrolyze a synthetic substrate (Boc-Leu-Gly-Arg-pNA) in the presence of Pro-CE, activation of ProCE (i.e., formation of CE) was determined.
[0195]Through the same experiment as carried out in Example 1, the activity of recombinant factor B was confirmed. In the study of reactivity of Et, the highest Et reactivity was attained in a sample (MOI=0.1, 96 h). Thus, the sample was employed for reconstitution of the factor C-cascade (Table 5 and FIG. 9).
TABLE-US-00005 TABLE 5 [Endpoint Assay] Reactivity (A 405 to Sample Sample No. 492 nm) Average Endotoxin Recombinant 1) MOI = 0.02, 48 h 0.022 -- 0.022 (0 ng/ Factor B 2) MOI = 0.02, 72 h 0.022 -- 0.022 mL) (×5 diluted) 3) MOI = 0.02, 96 h 0.022 -- 0.022 4) MOI = 0.1, 48 h 0.023 -- 0.023 5) MOI = 0.1, 72 h 0.018 -- 0.018 6) MOI = 0.1, 96 h 0.018 -- 0.018 7) MOI = 0.5, 48 h 0.010 -- 0.010 8) MOI = 0.5, 72 h 0.019 -- 0.019 9) MOI = 0.5, 96 h 0.018 -- 0.018 (×5 diluted) 10) Non-infected 0.016 -- 0.016 cells 11) Wild-type- 0.016 -- 0.016 infected Endotoxin Recombinant 1) MOI = 0.02, 48 h 0.044 0.043 0.044 (10 ng/ Factor B 2) MOI = 0.02, 72 h 0.232 0.230 0.231 mL) (×5 diluted) 3) MOI = 0.02, 96 h 0.440 0.451 0.446 4) MOI = 0.1, 48 h 0.340 0.318 0.329 5) MOI = 0.1, 72 h 0.568 0.572 0.570 6) MOI = 0.1, 96 h 0.617 0.622 0.620 7) MOI = 0.5, 48 h 0.300 0.276 0.288 8) MOI = 0.5, 72 h 0.478 0.462 0.470 9) MOI = 0.5, 96 h 0.368 0.369 0.369 (×5 diluted) 10) Non-infected 0.034 0.033 0.034 cells 11) Wild-type- 0.035 0.034 0.035 infected
[0196]In order to confirm whether or not cascade reaction proceeds when a different limulus-derived factor is employed in combination, recombinant factor C (derived from Tachypleus rotundicauda), which is a commercial element of PyroGene (product of Cambrex) was used.
Reagents
[0197](1) recombinant factor B (culture supernatant, MOI=0.1, 96 h)(2) recombinant Pro-CE (culture supernatant of sample 2 in Example 2)(3) recombinant factor C (PyroGene (commercial product), non-diluted)(4) Et: E. coli O111:B4-derived (Product of Sigma) was processed with distilled water to 1 mg/mL, followed by ×10-stepwise dilution
[0198]In order to confirm that the experimental results are attributable to reconstitution of the cascade, the following combination samples were tested.
Sample A: recombinant Pro-CE, recombinant factor B, and recombinant factor CSample B: recombinant Pro-CE, recombinant factor B, and distilled waterSample C: recombinant Pro-CE, recombinant factor C, and distilled waterSample D: recombinant factor B, recombinant factor C, and distilled waterSample E: recombinant Pro-CE and distilled waterSample F: recombinant factor B and distilled waterSample G: recombinant factor C and distilled water
[0199]The aforementioned culture supernatants (1) and (2) employed in the samples each were diluted in advance five-fold with ice-cooled Tris-HCl buffer (pH: 7.5) (50 mL) containing 150 mM NaCl. The total volume of each sample was adjusted to 60 μL.
[0200]To each sample, there were added Tris-HCl buffer (pH: 8.0) (final concentration: 80 mM), MgSO4 (final concentration: 64 mM), CaCl2 (final concentration: 0.4 mM), Na2SO4 (final concentration: 8 mM), dextran (final concentration: 2.4%), distilled water for injection (15 μL), Boc-Leu-Gly-Arg-pNA substrate (final concentration: 0.24 mM), and Et solution (0 or 100 ng/mL) (25 μL). The total volume of the sample was adjusted to 125 μL, and the sample was transferred to a Wellreader SK603, where it was allowed to react at 37° C. for 10 hours. The absorbance of the sample was automatically determined at a measurement wavelength of 405 nm (control wavelength: 492 nm). The measurement was carried out twice, and the average absorbance was calculated. The results are shown in Table 6 and FIG. 10.
TABLE-US-00006 TABLE 6 Reactivity Et concentration Reconstitution (A405 to 492 nm) (ng/mL) system Av. 0 (A) 0.024 0.022 0.023 PCE + FB + FC (B) PCE + FB 0.023 0.022 0.023 (C) PCE + FC 0.020 0.020 0.020 (D) FB + FC 0.024 0.025 0.025 (E) PCE 0.009 0.006 0.008 (F) FB 0.022 0.022 0.022 (G) FC 0.022 0.023 0.023 100 (A) 0.652 0.645 0.649 PCE + FB + FC (B) PCE + FB 0.022 0.023 0.023 (C) PCE + FC 0.029 0.030 0.030 (D) FB + FC 0.032 0.034 0.033 (E) PCE 0.006 0.006 0.006 (F) FB 0.024 0.026 0.025 (G) FC 0.034 0.035 0.035
[0201]The hydrolysis performance of the factors (samples A, B, and E to G) with respect to the Boc-Leu-Gly-Arg-pNA substrate in the presence of Et at high concentration was investigated. As a result, in the samples containing recombinant Pro-CE and/or recombinant factor B, activation attributed to high concentration Et was not observed. In contrast, remarkable hydrolysis activity was observed in a sample containing recombinant factor C and in the complete reconstitution system containing recombinant factors C and B and recombinant Pro-CE. However, the hydrolysis activity induced in the complete reconstitution system containing recombinant Pro-CE, recombinant B, and recombinant C was remarkably high (about some 103) as compared with that induced in the sample containing only recombinant factor C (FIG. 11).
[0202]As is clear from Table 6 and FIGS. 10 and 11, similar to the case of a native factor contained in amebocyte lysate (native factor LAL), cascade reaction of Et proceeds in a concentration-dependent manner through employment of recombinant factors C and B and recombinant Pro-CE in combination. Also, even when a different limulus-derived clotting factor is employed for reconstitution of the cascade, the same enzymatic activity as that of native factor LAL was found to be expressed.
Example 5
Effect of Metal Salt on Et Reaction Activity of a Complete Reconstitution System
[0203]In Et reaction in the complete reconstitution system (sample A) shown in Example 4, effects of variation amount of metal salt on Et reaction activity was investigated.
(A) Effect of Magnesium Sulfate (No Calcium Chloride or Sodium Chloride was Added)
(a) In a Concentration Range of 0 to 100 mM
[0204]In Example 5, the magnesium sulfate concentration in Et reaction in the complete reconstitution system of Example 4, which was 64 mM, was changed within the range of 0 to 100 mM, and the change in reaction activity was observed (during reaction). The results are shown in Table 7 and FIG. 12. In the reaction, an Et solution of 0 ng/mL (control) and that of 100 ng/mL were employed.
TABLE-US-00007 TABLE 7 NaCl Et concentration Reactivity concentration (mM: during (mAbs/min) (ng/mL) reaction) Av. 0 0 0.21 -- 0.21 6.25 0.22 -- 0.22 12.5 0.21 -- 0.21 25 0.21 -- 0.21 50 0.20 -- 0.20 100 0.19 -- 0.19 200 0.14 -- 0.14 20 0 27.07 27.50 27.29 6.25 25.83 25.79 25.81 12.5 28.30 29.32 28.81 25 28.18 28.65 28.42 50 27.27 27.96 27.62 100 21.79 21.46 21.63 200 17.66 18.42 18.04
[0205]As is clear from Table 7 and FIG. 12, in the complete reconstitution system of the method 1 of the present invention, the reactivity value significantly decreased in response to the increase in concentration of magnesium sulfate added to the measurement system.
(b) In a Concentration Range of 0 to 10 mM
[0206]In the reaction system (a) of the Example (Et concentration: 0 ng/mL (control) or 20 ng/mL), the magnesium sulfate concentration was changed to 0 to 10 mM during reaction, and change in activity was observed after reconstitution. The results are shown in Table 8 and FIG. 13.
TABLE-US-00008 TABLE 8 MgSO4 Et concentration Reactivity concentration (mM: during (mAbs/min) (ng/mL) reaction) Av. 0 0 0.11 -- 0.11 1 0.10 -- 0.10 2 0.08 -- 0.08 4 0.08 -- 0.08 6 0.09 -- 0.09 8 0.08 -- 0.08 10 0.09 -- 0.09 20 0 24.03 23.96 24.00 1 22.33 22.42 22.38 2 21.53 22.88 22.21 4 20.22 19.59 19.91 6 18.70 18.40 18.55 8 15.33 15.78 15.56 10 14.44 14.57 14.51
[0207]As is clear from Table 8 and FIG. 13, in the complete reconstitution system of the method 1 of the present invention, even when the magnesium sulfate concentration was 10 mM or lower, Et reaction was suppressed in a concentration-dependent manner.
[0208]Thus, in Et reaction in the measurement system, activity of the factors can be clearly detected and maintained after reconstitution, even when no magnesium sulfate was added.
(B) Effects of Calcium Chloride (No Magnesium Sulfate or Sodium Chloride was Added)
[0209]In Et reaction in the complete reconstitution system of Example 4 (Et concentration: 0 (control) or 20 ng/mL), the calcium chloride concentration was changed to 0 to 5 mM during reaction, and change in activity was observed after reconstitution. The results are shown in Table 9 and FIG. 14.
TABLE-US-00009 TABLE 9 CaCl2 Et concentration Reactivity concentration (mM: during (mAbs/min) (ng/mL) reaction) Av. 0 0 0.06 -- 0.06 0.5 0.03 -- 0.03 1 0.13 -- 0.13 2 0.36 -- 0.36 3 0.64 -- 0.64 4 0.73 -- 0.73 5 0.71 -- 0.71 20 0 30.83 31.02 30.93 0.5 21.66 22.79 22.23 1 17.53 20.01 18.77 2 14.09 14.60 14.35 3 11.78 12.96 12.37 4 9.88 10.04 9.96 5 9.15 10.04 9.60 (Slightly turbid at a CaCl2 concentration of 1 to 5 mM)
[0210]As is clear from Table 9 and FIG. 14, in Et reaction in the complete reconstitution system, activity of the factors can be clearly detected and maintained after reconstitution, even when no calcium chloride was added. The feature is similar to the case of addition of a magnesium salt.
(C) Effects of Sodium Chloride (No Magnesium Sulfate or Calcium Chloride was Added)
[0211]In the experiment system, Cl.sup.- was present at a concentration of 80 mM in reaction buffer (Tris-Cl, pH=8.0) during reaction. However, since Na.sup.+ was absent in the experiment system, the buffer-derived Cl.sup.- was not included in the sodium chloride concentration.
[0212]In Et reaction in the complete reconstitution system of Example 4, Et concentration was 0 (control) or 20 ng/mL). In Example 5, the calcium chloride concentration, which was 150 mm in Example 4, was changed in the range of 0 to 2.5 M (0, 0.078, 0.156, 0.313, 0.625, 1.25, and 2.5 (M)), and change in reactivity value was observed (during reaction). The results are shown in Table 10 and FIG. 15.
TABLE-US-00010 TABLE 10 NaCl Et concentration Reactivity concentration (mM: during (mAbs/min) (ng/mL) reaction) Av. 0 0 0.21 -- 0.21 6.25 0.22 -- 0.22 12.5 0.21 -- 0.21 25 0.21 -- 0.21 50 0.20 -- 0.20 100 0.19 -- 0.19 200 0.14 -- 0.14 20 0 27.07 27.50 27.29 6.25 25.83 25.79 25.81 12.5 28.30 29.32 28.81 25 28.18 28.65 28.42 50 27.27 27.96 27.62 100 21.79 21.46 21.63 200 17.66 18.42 18.04
[0213]As is clear from Table 10 and FIG. 15, when the sodium chloride concentration during reaction was 0 to 50 mm, virtually no effect was observed on activity after reconstitution. However, when the concentration was higher, reaction was significantly suppressed (reduction by about 30% at 200 mM).
Example 6
Substrate Specificity of Et Reaction in the Complete Reconstitution System
[0214]In Example 6, a synthetic chromogenic substrate Boc-Val-Pro-Arg-pNA (Boc-VPR-pNA) (acetate salt) (hereinafter referred to as substrate 1)--similar to a synthetic fluorescent substrate contained in an Et assay reagent (PyroGene, product of Cambrex) containing recombinant factor C as an essential component (Boc-Val-Pro-Arg-MCA)--was employed. Comparison in terms of Et reaction in the complete reconstitution system of Example 4 was made to Boc-Leu-Gly-Arg-pNA (Boc-LGR-pNA) (hereinafter referred to as substrate 2), which is an optimum substrate in conventional LAL reaction.
[0215]The each substrate (1 or 2) concentration in Et reaction was predetermined to 0.3 mM.
[0216]Table 11 and FIG. 16 show the results.
TABLE-US-00011 TABLE 11 Et Reactivity concentration (mAbs/min) Substrate (EU/mL) Av. Boc-LGR- 0 0.10 0.09 0.10 pNA 0.1 0.47 0.45 0.46 1 3.87 3.76 3.82 10 28.61 27.90 28.26 100 50.71 52.37 51.54 1000 70.48 70.23 70.36 Boc-VPR- 0 0.11 0.09 0.10 pNA 0.1 0.12 0.11 0.12 1 0.12 0.14 0.13 10 0.45 0.48 0.47 100 3.54 3.34 3.44 1000 15.46 15.30 15.38
[0217]As is clear from Table 11 and FIG. 16, Et activity in the complete reconstitution system is about 200 times higher in the case of Boc-Leu-Gly-Arg-pNA (substrate 2) than that in the case of Boc-Val-Pro-Arg-pNA (substrate 1).
[0218]In an additional experiment, Et reaction in the system only containing recombinant factor C (substrate concentration: 0.3 mM (substrates 1 and 2) was investigated. As a result, reactivity value when substrate 1 was used was about 1.6 times higher than that when substrate 2 was used.
[0219]Therefore, as compared with use of recombinant factor C alone, a complete reconstitution system exhibits remarkably high reactivity (Et activity). Those skilled in the art readily understand that the same tendency may be observed when Et is changed to (1→3)-β-D-glucan. Thus, according to the present invention, detection and determination of endotoxin and (1→3)-β-D-glucan at high sensitivity and high reproducibility can be realized, even when the assay target is present in a small amount.
INDUSTRIAL APPLICABILITY
[0220]The present invention provides an in vitro tool and a method for genetically mass-producing a limulus-derived Pro-CE or for detecting and determining a bacterial component derived from a microorganism at high efficiency and reproducibility. The Pro-CE produced according to the present invention serves as a main factor forming, with another recombinant factor relating to LAL reaction, a reaction system. On the basis of detection and assay of an Et and BG, the method of the present invention finds a wide range of non-temporary uses, safety evaluation of pharmaceutical products and medical tools; serum diagnosis of sepsis, fungal infections, etc.; tools for detecting microorganism contamination in environmental and food hygiene fields; reagents for use in research fields.
Sequence CWU
1
2011501DNATachypleus tridentatusCDS(217)..(1344) 1gcaatacagg ctacaaacat
ctcatcagac gcattacctg gttgtttaat tccgcgaaag 60cttcacgaga acaagtcaaa
cttagcttgt ggtcccgcga ctgactgcct ggaaagtagt 120tcccaaaatt gcggctttat
aaatcaacct aaacaaaacc gtcctgaaac tttaccgttc 180cacatccacc gaggtagccg
ggtcgtcctc agcagt atg ttg gtg aat aac gtg 234Met Leu Val Asn Asn
Val1 5ttt tca cta ctg tgt ttc cca ctc ttg atg tct gtg gtt
aga tgc agt 282Phe Ser Leu Leu Cys Phe Pro Leu Leu Met Ser Val Val
Arg Cys Ser10 15 20act ctc agc aga cag
cgt aga cag ttt gtt ttc cct gac gag gaa gaa 330Thr Leu Ser Arg Gln
Arg Arg Gln Phe Val Phe Pro Asp Glu Glu Glu25 30
35ctt tgc tca aac cga ttt act gaa gaa gga aca tgc aaa aat gtc
ttg 378Leu Cys Ser Asn Arg Phe Thr Glu Glu Gly Thr Cys Lys Asn Val
Leu40 45 50gat tgt aga ata ctt tta caa
aaa aat gat tat aat tta ctc aaa gaa 426Asp Cys Arg Ile Leu Leu Gln
Lys Asn Asp Tyr Asn Leu Leu Lys Glu55 60
65 70tca ata tgc ggc ttt gaa ggc ata aca ccc aaa gtt
tgt tgt ccg aaa 474Ser Ile Cys Gly Phe Glu Gly Ile Thr Pro Lys Val
Cys Cys Pro Lys75 80 85tca agc cat gta
att tca agt aca cag gca cct cca gaa acc act acg 522Ser Ser His Val
Ile Ser Ser Thr Gln Ala Pro Pro Glu Thr Thr Thr90 95
100act gaa cgc cca cca aaa cag ata cca ccc aat ctt cct gaa
gtg tgt 570Thr Glu Arg Pro Pro Lys Gln Ile Pro Pro Asn Leu Pro Glu
Val Cys105 110 115gga att cac aat act aca
act acc agg att att gga ggt cgg gaa gca 618Gly Ile His Asn Thr Thr
Thr Thr Arg Ile Ile Gly Gly Arg Glu Ala120 125
130cct att gga gcc tgg ccg tgg atg act gct gtc tac ata aaa caa gga
666Pro Ile Gly Ala Trp Pro Trp Met Thr Ala Val Tyr Ile Lys Gln Gly135
140 145 150gga atc aga agt
gtt cag tgt ggt ggc gca ctt gtc act aac agg cac 714Gly Ile Arg Ser
Val Gln Cys Gly Gly Ala Leu Val Thr Asn Arg His155 160
165gtg att aca gct tcg cac tgt gtt gta aac agt gca gga aca
gat gtg 762Val Ile Thr Ala Ser His Cys Val Val Asn Ser Ala Gly Thr
Asp Val170 175 180atg cca gct gat gta ttc
tcg gtt cgt ctg ggt gaa cac aat tta tac 810Met Pro Ala Asp Val Phe
Ser Val Arg Leu Gly Glu His Asn Leu Tyr185 190
195agt acc gat gac gat tcg aat cca ata gat ttt gca gtt acg tcg gtg
858Ser Thr Asp Asp Asp Ser Asn Pro Ile Asp Phe Ala Val Thr Ser Val200
205 210aaa cat cac gaa cac ttt gta ctc gcg
acg tat ttg aat gac atc gca 906Lys His His Glu His Phe Val Leu Ala
Thr Tyr Leu Asn Asp Ile Ala215 220 225
230att cta acg tta aat gac aca gtt acg ttt aca gac aga att
cga ccc 954Ile Leu Thr Leu Asn Asp Thr Val Thr Phe Thr Asp Arg Ile
Arg Pro235 240 245att tgt cta cct tat cgt
aag ttg aga tac gat gat cta gca atg aga 1002Ile Cys Leu Pro Tyr Arg
Lys Leu Arg Tyr Asp Asp Leu Ala Met Arg250 255
260aaa ccg ttt atc act gga tgg gga aca aca gca ttt aac ggc cca tct
1050Lys Pro Phe Ile Thr Gly Trp Gly Thr Thr Ala Phe Asn Gly Pro Ser265
270 275agt gca gtg ttg aga gaa gta cag tta
cca ata tgg gaa cac gag gcc 1098Ser Ala Val Leu Arg Glu Val Gln Leu
Pro Ile Trp Glu His Glu Ala280 285 290tgt
aga cag gcc tac gag aag gat tta aat att aca aac gtg tat atg 1146Cys
Arg Gln Ala Tyr Glu Lys Asp Leu Asn Ile Thr Asn Val Tyr Met295
300 305 310tgt gct ggc ttt gca gat
ggc ggg aag gat gct tgc cag ggt gat tct 1194Cys Ala Gly Phe Ala Asp
Gly Gly Lys Asp Ala Cys Gln Gly Asp Ser315 320
325gga ggt cca atg atg ttg cct gtt aaa acc gga gag ttt tat ctc att
1242Gly Gly Pro Met Met Leu Pro Val Lys Thr Gly Glu Phe Tyr Leu Ile330
335 340gga att gtg tct ttc gga aag aaa tgc
gca ttg cct gga ttt cct ggg 1290Gly Ile Val Ser Phe Gly Lys Lys Cys
Ala Leu Pro Gly Phe Pro Gly345 350 355gtt
tac aca aaa gtg aca gag ttt tta gat tgg att gca gaa cat atg 1338Val
Tyr Thr Lys Val Thr Glu Phe Leu Asp Trp Ile Ala Glu His Met360
365 370gtg tag actaaagctg tgaatgactc catttcgaat
atttaaccca tactcgcctg 1394Val375tagcaaaacg actgaagaat caagcagggg
acaaaaccaa cttttttttt tttttttctt 1454caatagttct accatgtatt gaaaaccaac
ataagacaat caaataa 15012375PRTTachypleus tridentatus 2Met
Leu Val Asn Asn Val Phe Ser Leu Leu Cys Phe Pro Leu Leu Met1
5 10 15Ser Val Val Arg Cys Ser Thr
Leu Ser Arg Gln Arg Arg Gln Phe Val20 25
30Phe Pro Asp Glu Glu Glu Leu Cys Ser Asn Arg Phe Thr Glu Glu Gly35
40 45Thr Cys Lys Asn Val Leu Asp Cys Arg Ile
Leu Leu Gln Lys Asn Asp50 55 60Tyr Asn
Leu Leu Lys Glu Ser Ile Cys Gly Phe Glu Gly Ile Thr Pro65
70 75 80Lys Val Cys Cys Pro Lys Ser
Ser His Val Ile Ser Ser Thr Gln Ala85 90
95Pro Pro Glu Thr Thr Thr Thr Glu Arg Pro Pro Lys Gln Ile Pro Pro100
105 110Asn Leu Pro Glu Val Cys Gly Ile His
Asn Thr Thr Thr Thr Arg Ile115 120 125Ile
Gly Gly Arg Glu Ala Pro Ile Gly Ala Trp Pro Trp Met Thr Ala130
135 140Val Tyr Ile Lys Gln Gly Gly Ile Arg Ser Val
Gln Cys Gly Gly Ala145 150 155
160Leu Val Thr Asn Arg His Val Ile Thr Ala Ser His Cys Val Val
Asn165 170 175Ser Ala Gly Thr Asp Val Met
Pro Ala Asp Val Phe Ser Val Arg Leu180 185
190Gly Glu His Asn Leu Tyr Ser Thr Asp Asp Asp Ser Asn Pro Ile Asp195
200 205Phe Ala Val Thr Ser Val Lys His His
Glu His Phe Val Leu Ala Thr210 215 220Tyr
Leu Asn Asp Ile Ala Ile Leu Thr Leu Asn Asp Thr Val Thr Phe225
230 235 240Thr Asp Arg Ile Arg Pro
Ile Cys Leu Pro Tyr Arg Lys Leu Arg Tyr245 250
255Asp Asp Leu Ala Met Arg Lys Pro Phe Ile Thr Gly Trp Gly Thr
Thr260 265 270Ala Phe Asn Gly Pro Ser Ser
Ala Val Leu Arg Glu Val Gln Leu Pro275 280
285Ile Trp Glu His Glu Ala Cys Arg Gln Ala Tyr Glu Lys Asp Leu Asn290
295 300Ile Thr Asn Val Tyr Met Cys Ala Gly
Phe Ala Asp Gly Gly Lys Asp305 310 315
320Ala Cys Gln Gly Asp Ser Gly Gly Pro Met Met Leu Pro Val
Lys Thr325 330 335Gly Glu Phe Tyr Leu Ile
Gly Ile Val Ser Phe Gly Lys Lys Cys Ala340 345
350Leu Pro Gly Phe Pro Gly Val Tyr Thr Lys Val Thr Glu Phe Leu
Asp355 360 365Trp Ile Ala Glu His Met
Val370 37531146DNATachypleus tridentatusCDS(1)..(1143)
3atg ttg gtg aat aac gtg ttt tca cta ctg tgt ttc cca ctc ttg atg
48Met Leu Val Asn Asn Val Phe Ser Leu Leu Cys Phe Pro Leu Leu Met1
5 10 15tct gtg gtt aga tgc agt
act ctc agc aga cag cgt aga cag ttt gtt 96Ser Val Val Arg Cys Ser
Thr Leu Ser Arg Gln Arg Arg Gln Phe Val20 25
30ttc cct gac gag gaa gaa ctt tgc tca aac cga ttt act gaa gaa gga
144Phe Pro Asp Glu Glu Glu Leu Cys Ser Asn Arg Phe Thr Glu Glu Gly35
40 45aca tgc aaa aat gtc ttg gat tgt aga
ata ctt tta caa aaa aat gat 192Thr Cys Lys Asn Val Leu Asp Cys Arg
Ile Leu Leu Gln Lys Asn Asp50 55 60tat
aat tta ctc aaa gaa tca ata tgc ggc ttt gaa ggc ata aca ccc 240Tyr
Asn Leu Leu Lys Glu Ser Ile Cys Gly Phe Glu Gly Ile Thr Pro65
70 75 80aaa gtt tgt tgt ccg aaa
tca agc cat gta att tca agt aca cag gca 288Lys Val Cys Cys Pro Lys
Ser Ser His Val Ile Ser Ser Thr Gln Ala85 90
95cct cca gaa acc act acg act gaa cgc cca cca aaa cag ata cca ccc
336Pro Pro Glu Thr Thr Thr Thr Glu Arg Pro Pro Lys Gln Ile Pro Pro100
105 110aat ctt cct gaa gtg tgt gga att cac
aat act aca act acc agg att 384Asn Leu Pro Glu Val Cys Gly Ile His
Asn Thr Thr Thr Thr Arg Ile115 120 125att
gga ggt cgg gaa gca cct att gga gcc tgg ccg tgg atg act gct 432Ile
Gly Gly Arg Glu Ala Pro Ile Gly Ala Trp Pro Trp Met Thr Ala130
135 140gtc tac ata aaa caa gga gga atc aga agt gtt
cag tgt ggt ggc gca 480Val Tyr Ile Lys Gln Gly Gly Ile Arg Ser Val
Gln Cys Gly Gly Ala145 150 155
160ctt gtc act aac agg cac gtg att aca gct tcg cac tgt gtt gta aac
528Leu Val Thr Asn Arg His Val Ile Thr Ala Ser His Cys Val Val Asn165
170 175agt gca gga aca gat gtg atg cca gct
gat gta ttc tcg gtt cgt ctg 576Ser Ala Gly Thr Asp Val Met Pro Ala
Asp Val Phe Ser Val Arg Leu180 185 190ggt
gaa cac aat tta tac agt acc gat gac gat tcg aat cca ata gat 624Gly
Glu His Asn Leu Tyr Ser Thr Asp Asp Asp Ser Asn Pro Ile Asp195
200 205ttt gca gtt acg tcg gtg aaa cat cac gaa cac
ttt gta ctc gcg acg 672Phe Ala Val Thr Ser Val Lys His His Glu His
Phe Val Leu Ala Thr210 215 220tat ttg aat
gac atc gca att cta acg tta aat gac aca gtt acg ttt 720Tyr Leu Asn
Asp Ile Ala Ile Leu Thr Leu Asn Asp Thr Val Thr Phe225
230 235 240aca gac aga att cga ccc att
tgt cta cct tat cgt aag ttg aga tac 768Thr Asp Arg Ile Arg Pro Ile
Cys Leu Pro Tyr Arg Lys Leu Arg Tyr245 250
255gat gat cta gca atg aga aaa ccg ttt atc act gga tgg gga aca aca
816Asp Asp Leu Ala Met Arg Lys Pro Phe Ile Thr Gly Trp Gly Thr Thr260
265 270gca ttt aac ggc cca tct agt gca gtg
ttg aga gaa gta cag tta cca 864Ala Phe Asn Gly Pro Ser Ser Ala Val
Leu Arg Glu Val Gln Leu Pro275 280 285ata
tgg gaa cac gag gcc tgt aga cag gcc tac gag aag gat tta aat 912Ile
Trp Glu His Glu Ala Cys Arg Gln Ala Tyr Glu Lys Asp Leu Asn290
295 300att aca aac gtg tat atg tgt gct ggc ttt gca
gat ggc ggg aag gat 960Ile Thr Asn Val Tyr Met Cys Ala Gly Phe Ala
Asp Gly Gly Lys Asp305 310 315
320gct tgc cag ggt gat tct gga ggt cca atg atg ttg cct gtt aaa acc
1008Ala Cys Gln Gly Asp Ser Gly Gly Pro Met Met Leu Pro Val Lys Thr325
330 335gga gag ttt tat ctc att gga att gtg
tct ttc gga aag aaa tgc gca 1056Gly Glu Phe Tyr Leu Ile Gly Ile Val
Ser Phe Gly Lys Lys Cys Ala340 345 350ttg
cct gga ttt cct ggg gtt tac aca aaa gtg aca gag ttt tta gat 1104Leu
Pro Gly Phe Pro Gly Val Tyr Thr Lys Val Thr Glu Phe Leu Asp355
360 365tgg att gca gaa cat atg gtg cat cac cat cac
cat cac tag 1146Trp Ile Ala Glu His Met Val His His His His
His His370 375 3804381PRTTachypleus
tridentatus 4Met Leu Val Asn Asn Val Phe Ser Leu Leu Cys Phe Pro Leu Leu
Met1 5 10 15Ser Val Val
Arg Cys Ser Thr Leu Ser Arg Gln Arg Arg Gln Phe Val20 25
30Phe Pro Asp Glu Glu Glu Leu Cys Ser Asn Arg Phe Thr
Glu Glu Gly35 40 45Thr Cys Lys Asn Val
Leu Asp Cys Arg Ile Leu Leu Gln Lys Asn Asp50 55
60Tyr Asn Leu Leu Lys Glu Ser Ile Cys Gly Phe Glu Gly Ile Thr
Pro65 70 75 80Lys Val
Cys Cys Pro Lys Ser Ser His Val Ile Ser Ser Thr Gln Ala85
90 95Pro Pro Glu Thr Thr Thr Thr Glu Arg Pro Pro Lys
Gln Ile Pro Pro100 105 110Asn Leu Pro Glu
Val Cys Gly Ile His Asn Thr Thr Thr Thr Arg Ile115 120
125Ile Gly Gly Arg Glu Ala Pro Ile Gly Ala Trp Pro Trp Met
Thr Ala130 135 140Val Tyr Ile Lys Gln Gly
Gly Ile Arg Ser Val Gln Cys Gly Gly Ala145 150
155 160Leu Val Thr Asn Arg His Val Ile Thr Ala Ser
His Cys Val Val Asn165 170 175Ser Ala Gly
Thr Asp Val Met Pro Ala Asp Val Phe Ser Val Arg Leu180
185 190Gly Glu His Asn Leu Tyr Ser Thr Asp Asp Asp Ser
Asn Pro Ile Asp195 200 205Phe Ala Val Thr
Ser Val Lys His His Glu His Phe Val Leu Ala Thr210 215
220Tyr Leu Asn Asp Ile Ala Ile Leu Thr Leu Asn Asp Thr Val
Thr Phe225 230 235 240Thr
Asp Arg Ile Arg Pro Ile Cys Leu Pro Tyr Arg Lys Leu Arg Tyr245
250 255Asp Asp Leu Ala Met Arg Lys Pro Phe Ile Thr
Gly Trp Gly Thr Thr260 265 270Ala Phe Asn
Gly Pro Ser Ser Ala Val Leu Arg Glu Val Gln Leu Pro275
280 285Ile Trp Glu His Glu Ala Cys Arg Gln Ala Tyr Glu
Lys Asp Leu Asn290 295 300Ile Thr Asn Val
Tyr Met Cys Ala Gly Phe Ala Asp Gly Gly Lys Asp305 310
315 320Ala Cys Gln Gly Asp Ser Gly Gly Pro
Met Met Leu Pro Val Lys Thr325 330 335Gly
Glu Phe Tyr Leu Ile Gly Ile Val Ser Phe Gly Lys Lys Cys Ala340
345 350Leu Pro Gly Phe Pro Gly Val Tyr Thr Lys Val
Thr Glu Phe Leu Asp355 360 365Trp Ile Ala
Glu His Met Val His His His His His His370 375
380526DNAArtificialPrimer for sequencing 5tctagaatgt tggtgaataa
cgtgtt 26640DNAArtificialPrimer
for sequencing 6agatctagtg atggtgatgg tgatgcacca tatgttctgc
40718DNAArtificialPrimer for sequencing 7tcacaaactg gaaatgtc
18818DNAArtificialPrimer
for sequencing 8ccggaccagt gaacagag
18920DNAArtificialPrimer for sequencing 9aatcagaagt
gttcagtgtg
201020DNAArtificialPrimer for sequencing 10tcattcaaat acgtcgcgag
201121DNAArtificialPCR Primer
11gccattgtaa tgagacgcac a
211223DNAArtificialPCR Primer 12cgtacaacaa ttgtctgtaa atc
231321DNAArtificialPrimer for sequencing
13tagttgctga tatcatggag a
211418DNAArtificialPrimer for sequencing 14tcacaaactg gaaatgtc
18151887DNATachypleus
tridentatusCDS(143)..(1345) 15gttgaatctt accggtaaga attagataac aaatttttaa
aagttacact aacagaagct 60ttttgacgtc agtatcttat atgtattacg tattcccaat
ttaattggat cgttaacgcc 120agacaactaa ccattttctg ga atg acg tgg ata tgt
gtg ata acg ttg ttt 172Met Thr Trp Ile Cys Val Ile Thr Leu Phe1
5 10gct ctg gct tct gct acg ttg ggt aac aaa gtt
agt aga gtg ggg gtc 220Ala Leu Ala Ser Ala Thr Leu Gly Asn Lys Val
Ser Arg Val Gly Val15 20 25ctc ttc ccc
aag aca cgg aac gac aat gag tgt aca gca aga ggg gga 268Leu Phe Pro
Lys Thr Arg Asn Asp Asn Glu Cys Thr Ala Arg Gly Gly30 35
40ttg aaa gga tcc tgc aaa tcc ctc ata gac tgt cct agt
gtc ttg gct 316Leu Lys Gly Ser Cys Lys Ser Leu Ile Asp Cys Pro Ser
Val Leu Ala45 50 55acg ttg aag gac agt
ttt cct gtc gtt tgc tct tgg aat ggt cga ttt 364Thr Leu Lys Asp Ser
Phe Pro Val Val Cys Ser Trp Asn Gly Arg Phe60 65
70cag cct att gtc tgc tgt cct gat gca ata gca cca cca cct gta
acc 412Gln Pro Ile Val Cys Cys Pro Asp Ala Ile Ala Pro Pro Pro Val
Thr75 80 85 90aca aca
gct gta act gta ata tct aca aaa gaa cca aag ctt cca aga 460Thr Thr
Ala Val Thr Val Ile Ser Thr Lys Glu Pro Lys Leu Pro Arg95
100 105tta cat ata tca ggt tgt gga aaa aga aaa gtc aaa
ata gat att aca 508Leu His Ile Ser Gly Cys Gly Lys Arg Lys Val Lys
Ile Asp Ile Thr110 115 120act gtt gga cgc
tct gga tca cca ata ctt cct ccg ata tct act cct 556Thr Val Gly Arg
Ser Gly Ser Pro Ile Leu Pro Pro Ile Ser Thr Pro125 130
135caa aat tca aca ggt ggg aga gga att att gct gga ggc gta
gaa gcc 604Gln Asn Ser Thr Gly Gly Arg Gly Ile Ile Ala Gly Gly Val
Glu Ala140 145 150aaa att ggc gcg tgg cct
tgg atg gca gct gtt ttt gtg aaa aac ttt 652Lys Ile Gly Ala Trp Pro
Trp Met Ala Ala Val Phe Val Lys Asn Phe155 160
165 170ggc att ggc aga ttc cac tgt gct ggt agc ata
atc agt aac aag tac 700Gly Ile Gly Arg Phe His Cys Ala Gly Ser Ile
Ile Ser Asn Lys Tyr175 180 185att ttg tca
gct gcc cac gcc ttc ctt atc gga ggt cga aag ttg acc 748Ile Leu Ser
Ala Ala His Ala Phe Leu Ile Gly Gly Arg Lys Leu Thr190
195 200cca act cgc tta gct gtc cgt gtg gga ggc cac tac
ata aag agg ggt 796Pro Thr Arg Leu Ala Val Arg Val Gly Gly His Tyr
Ile Lys Arg Gly205 210 215caa gag tat cca
gtg aaa gac gtg att atc cat cct cat tat gta gaa 844Gln Glu Tyr Pro
Val Lys Asp Val Ile Ile His Pro His Tyr Val Glu220 225
230aag gag aac tac aat gat ata gcc ata atc gag tta aaa gag
gaa ctg 892Lys Glu Asn Tyr Asn Asp Ile Ala Ile Ile Glu Leu Lys Glu
Glu Leu235 240 245 250aac
ttt acg gac ttg gtc aat cct ata tgt ctc cct gat cca gag aca 940Asn
Phe Thr Asp Leu Val Asn Pro Ile Cys Leu Pro Asp Pro Glu Thr255
260 265gta acg gat cca tta aaa gac aga att gtg act
gca gcg gga tgg ggc 988Val Thr Asp Pro Leu Lys Asp Arg Ile Val Thr
Ala Ala Gly Trp Gly270 275 280gat ctg gat
ttc tcc ggt cca cgg agc caa gtt cta cgt gag gta agc 1036Asp Leu Asp
Phe Ser Gly Pro Arg Ser Gln Val Leu Arg Glu Val Ser285
290 295atc cca gtt gtt cca gtt gat aaa tgt gat caa gcc
tat gag aaa ctc 1084Ile Pro Val Val Pro Val Asp Lys Cys Asp Gln Ala
Tyr Glu Lys Leu300 305 310aac acc cct tca
cta aaa aat ggg ata acg aat aac ttc ctt tgc gct 1132Asn Thr Pro Ser
Leu Lys Asn Gly Ile Thr Asn Asn Phe Leu Cys Ala315 320
325 330gga ttg gaa gaa gga ggg aaa gac gct
tgc caa ggc gat tct ggt gga 1180Gly Leu Glu Glu Gly Gly Lys Asp Ala
Cys Gln Gly Asp Ser Gly Gly335 340 345ccg
ttg atg cta gtg aac aac act agg tgg ata gta gta gga gtt gtg 1228Pro
Leu Met Leu Val Asn Asn Thr Arg Trp Ile Val Val Gly Val Val350
355 360tcg ttc ggg cac aag tgt gcc gag gaa gga tat
cct ggt gtg tac tcg 1276Ser Phe Gly His Lys Cys Ala Glu Glu Gly Tyr
Pro Gly Val Tyr Ser365 370 375cgc gta gcg
agt tac cta gac tgg atc gcg aaa gtt acg aac tcg tta 1324Arg Val Ala
Ser Tyr Leu Asp Trp Ile Ala Lys Val Thr Asn Ser Leu380
385 390gat cat gcc gtc act aac tga ttgtgcgtta aacaacatat
tttgttgtga 1375Asp His Ala Val Thr Asn395
400agaaatactg tccaaacgta acttttgaac ttgtattatg tattaacgta ttataagttc
1435tagttttggt ttcaaccact agatgacgca aatgctcgta ttgtccaaca gctttattcc
1495ttttgaagaa tattttttcg cacctacaag cttgtgaaac ttgtagataa tagttattcg
1555taattttctc attgttttta caatttttgt gtgatctgtt tttttatttt aactttgaag
1615taccggaatg aaaatattta attttatatt tcattaattc aaaacgtttt tgttctgggc
1675tgccatgtaa attgtataat aagtcgtggt tgtttagtaa gttatattat cggtagagtg
1735tacgttaaga tctgactgtg aattttgaag aaaaacctta catctaaaca ttttatcttt
1795actgactgta gttatttatc tctaatgttc agcgtgttta gccagtaatg taaatgtgaa
1855ttataattga ttaaagtttg attgaaatgt tt
188716400PRTTachypleus tridentatus 16Met Thr Trp Ile Cys Val Ile Thr Leu
Phe Ala Leu Ala Ser Ala Thr1 5 10
15Leu Gly Asn Lys Val Ser Arg Val Gly Val Leu Phe Pro Lys Thr
Arg20 25 30Asn Asp Asn Glu Cys Thr Ala
Arg Gly Gly Leu Lys Gly Ser Cys Lys35 40
45Ser Leu Ile Asp Cys Pro Ser Val Leu Ala Thr Leu Lys Asp Ser Phe50
55 60Pro Val Val Cys Ser Trp Asn Gly Arg Phe
Gln Pro Ile Val Cys Cys65 70 75
80Pro Asp Ala Ile Ala Pro Pro Pro Val Thr Thr Thr Ala Val Thr
Val85 90 95Ile Ser Thr Lys Glu Pro Lys
Leu Pro Arg Leu His Ile Ser Gly Cys100 105
110Gly Lys Arg Lys Val Lys Ile Asp Ile Thr Thr Val Gly Arg Ser Gly115
120 125Ser Pro Ile Leu Pro Pro Ile Ser Thr
Pro Gln Asn Ser Thr Gly Gly130 135 140Arg
Gly Ile Ile Ala Gly Gly Val Glu Ala Lys Ile Gly Ala Trp Pro145
150 155 160Trp Met Ala Ala Val Phe
Val Lys Asn Phe Gly Ile Gly Arg Phe His165 170
175Cys Ala Gly Ser Ile Ile Ser Asn Lys Tyr Ile Leu Ser Ala Ala
His180 185 190Ala Phe Leu Ile Gly Gly Arg
Lys Leu Thr Pro Thr Arg Leu Ala Val195 200
205Arg Val Gly Gly His Tyr Ile Lys Arg Gly Gln Glu Tyr Pro Val Lys210
215 220Asp Val Ile Ile His Pro His Tyr Val
Glu Lys Glu Asn Tyr Asn Asp225 230 235
240Ile Ala Ile Ile Glu Leu Lys Glu Glu Leu Asn Phe Thr Asp
Leu Val245 250 255Asn Pro Ile Cys Leu Pro
Asp Pro Glu Thr Val Thr Asp Pro Leu Lys260 265
270Asp Arg Ile Val Thr Ala Ala Gly Trp Gly Asp Leu Asp Phe Ser
Gly275 280 285Pro Arg Ser Gln Val Leu Arg
Glu Val Ser Ile Pro Val Val Pro Val290 295
300Asp Lys Cys Asp Gln Ala Tyr Glu Lys Leu Asn Thr Pro Ser Leu Lys305
310 315 320Asn Gly Ile Thr
Asn Asn Phe Leu Cys Ala Gly Leu Glu Glu Gly Gly325 330
335Lys Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Met Leu
Val Asn340 345 350Asn Thr Arg Trp Ile Val
Val Gly Val Val Ser Phe Gly His Lys Cys355 360
365Ala Glu Glu Gly Tyr Pro Gly Val Tyr Ser Arg Val Ala Ser Tyr
Leu370 375 380Asp Trp Ile Ala Lys Val Thr
Asn Ser Leu Asp His Ala Val Thr Asn385 390
395 400172394DNATachypleus tridentatusCDS(57)..(2078)
17gtggggggtt tagttgaaac agtaaatacg ttaactgttt aatcttgtta attgca atg
59Met1ttg gtg ttg ctg tgt tgt gtt gtt ttg cat gtt ggt gtt gca aga att
107Leu Val Leu Leu Cys Cys Val Val Leu His Val Gly Val Ala Arg Ile5
10 15tgc tgt agc cac gaa cca aag tgg cag
ctc gtc tgg tcg gat gaa ttt 155Cys Cys Ser His Glu Pro Lys Trp Gln
Leu Val Trp Ser Asp Glu Phe20 25 30acc
aat gga ata agt tct gat tgg gaa ttt gaa atg ggc aat ggc ctc 203Thr
Asn Gly Ile Ser Ser Asp Trp Glu Phe Glu Met Gly Asn Gly Leu35
40 45aat ggt tgg ggt aat aac gaa ctg caa tat tat
cgt cgt gaa aat gcc 251Asn Gly Trp Gly Asn Asn Glu Leu Gln Tyr Tyr
Arg Arg Glu Asn Ala50 55 60
65caa gtt gag gga ggg aaa ctg gta att act gct aaa aga gaa gac tat
299Gln Val Glu Gly Gly Lys Leu Val Ile Thr Ala Lys Arg Glu Asp Tyr70
75 80gat ggc ttc aaa tac act tct gct agg
ctg aaa acc cag ttt gat aaa 347Asp Gly Phe Lys Tyr Thr Ser Ala Arg
Leu Lys Thr Gln Phe Asp Lys85 90 95tct
tgg aag tat ggt aaa att gaa gcc aaa atg gcg att cca tca ttt 395Ser
Trp Lys Tyr Gly Lys Ile Glu Ala Lys Met Ala Ile Pro Ser Phe100
105 110cgg gga gtc tgg gtg atg ttc tgg atg tca gga
gac aac act aat tat 443Arg Gly Val Trp Val Met Phe Trp Met Ser Gly
Asp Asn Thr Asn Tyr115 120 125gtt aga tgg
cca tct tct ggt gaa att gac ttt att gaa cat aga aac 491Val Arg Trp
Pro Ser Ser Gly Glu Ile Asp Phe Ile Glu His Arg Asn130
135 140 145act aac aat gaa aaa gtc aga
gga act att cac tgg tcc act cct gac 539Thr Asn Asn Glu Lys Val Arg
Gly Thr Ile His Trp Ser Thr Pro Asp150 155
160ggt gct cat gcg cat cat aac aga gaa agt aat aca aat ggg att gat
587Gly Ala His Ala His His Asn Arg Glu Ser Asn Thr Asn Gly Ile Asp165
170 175tat cac att tat tct gta gag tgg aat
tct tcc att gtt aaa tgg ttt 635Tyr His Ile Tyr Ser Val Glu Trp Asn
Ser Ser Ile Val Lys Trp Phe180 185 190gtt
aat gga aat caa tac ttt gaa gtg aaa att cag gga gga gta aat 683Val
Asn Gly Asn Gln Tyr Phe Glu Val Lys Ile Gln Gly Gly Val Asn195
200 205ggg aaa agt gca ttt cgt aac aaa gtt ttc gtt
att tta aac atg gcg 731Gly Lys Ser Ala Phe Arg Asn Lys Val Phe Val
Ile Leu Asn Met Ala210 215 220
225att ggt gga aac tgg cca gga ttc gat gtt gct gac gag gct ttc cct
779Ile Gly Gly Asn Trp Pro Gly Phe Asp Val Ala Asp Glu Ala Phe Pro230
235 240gct aaa atg tac att gat tat gtc cgt
gta tac cag gat gcc agt aca 827Ala Lys Met Tyr Ile Asp Tyr Val Arg
Val Tyr Gln Asp Ala Ser Thr245 250 255tct
tct cct gtt ggg gat acc tct tta gat ggt tac tat ttt gtc caa 875Ser
Ser Pro Val Gly Asp Thr Ser Leu Asp Gly Tyr Tyr Phe Val Gln260
265 270aac agg cac agt gaa ttg tat ctt gat gtc act
gat gcc agt aac gaa 923Asn Arg His Ser Glu Leu Tyr Leu Asp Val Thr
Asp Ala Ser Asn Glu275 280 285gat gga gca
ttt ctg caa caa tgg tct tat agt ggt aat gag aac caa 971Asp Gly Ala
Phe Leu Gln Gln Trp Ser Tyr Ser Gly Asn Glu Asn Gln290
295 300 305cag ttt gat ttt gag cat ctc
gaa aat aat gtt tat aaa att act aat 1019Gln Phe Asp Phe Glu His Leu
Glu Asn Asn Val Tyr Lys Ile Thr Asn310 315
320aaa aaa agt gga aaa tct ttg gat gtt tat aat ttt ggg act gag aat
1067Lys Lys Ser Gly Lys Ser Leu Asp Val Tyr Asn Phe Gly Thr Glu Asn325
330 335ggt gtt aga atc caa cag tgg tca tat
gga ggg gct cgc aat cag cag 1115Gly Val Arg Ile Gln Gln Trp Ser Tyr
Gly Gly Ala Arg Asn Gln Gln340 345 350ttt
act gta caa agt gtt ggt gat ggt tat tat aag att att cca cgc 1163Phe
Thr Val Gln Ser Val Gly Asp Gly Tyr Tyr Lys Ile Ile Pro Arg355
360 365ggc agt gga aag tta gtg gaa gta gca gat ttt
agt aaa gat gca gga 1211Gly Ser Gly Lys Leu Val Glu Val Ala Asp Phe
Ser Lys Asp Ala Gly370 375 380
385ggg aag ata caa caa tgg tct gat aac aac caa tta tct gga cag tgg
1259Gly Lys Ile Gln Gln Trp Ser Asp Asn Asn Gln Leu Ser Gly Gln Trp390
395 400aaa ctt att aaa agt aaa agt tat tct
aaa tta att cag gca gaa agt 1307Lys Leu Ile Lys Ser Lys Ser Tyr Ser
Lys Leu Ile Gln Ala Glu Ser405 410 415tat
ttt gat tcc tca aaa gta caa ttg gaa gat acc tca gat gta gga 1355Tyr
Phe Asp Ser Ser Lys Val Gln Leu Glu Asp Thr Ser Asp Val Gly420
425 430ggt ggg aag aat gtt aaa tgt gat aat gaa gga
gcc tgg atg gct tat 1403Gly Gly Lys Asn Val Lys Cys Asp Asn Glu Gly
Ala Trp Met Ala Tyr435 440 445aag gat att
gat ttc ccc agt tca ggt aat tat cga ata gaa tac aga 1451Lys Asp Ile
Asp Phe Pro Ser Ser Gly Asn Tyr Arg Ile Glu Tyr Arg450
455 460 465gta gca agt gaa cgt gca gga
gga aag ctg tct ctg gat ttg aat gca 1499Val Ala Ser Glu Arg Ala Gly
Gly Lys Leu Ser Leu Asp Leu Asn Ala470 475
480ggc tct ata gtt ctt ggc atg ctg gat gtt cct tca aca gga gga tgg
1547Gly Ser Ile Val Leu Gly Met Leu Asp Val Pro Ser Thr Gly Gly Trp485
490 495cag aag tgg acc acc att tcc cat aca
gtg aat gtg gat tca ggt aca 1595Gln Lys Trp Thr Thr Ile Ser His Thr
Val Asn Val Asp Ser Gly Thr500 505 510tat
aac ttg ggg atc tat gtt caa cga gcc agc tgg aat atc aac tgg 1643Tyr
Asn Leu Gly Ile Tyr Val Gln Arg Ala Ser Trp Asn Ile Asn Trp515
520 525ata aag att aca aaa ata cct gaa cag tca aat
ttg aat caa ggg cgt 1691Ile Lys Ile Thr Lys Ile Pro Glu Gln Ser Asn
Leu Asn Gln Gly Arg530 535 540
545cgt aat tct aaa tta att cag gca gaa agt tat ttt agt tac tca gaa
1739Arg Asn Ser Lys Leu Ile Gln Ala Glu Ser Tyr Phe Ser Tyr Ser Glu550
555 560gta caa ctg gaa gat acc tta gat gta
gga ggt gga aag aat gtt aaa 1787Val Gln Leu Glu Asp Thr Leu Asp Val
Gly Gly Gly Lys Asn Val Lys565 570 575tgt
gat aaa gaa ggg gcc tgg atg gct tac aag gat att gat ttc ccc 1835Cys
Asp Lys Glu Gly Ala Trp Met Ala Tyr Lys Asp Ile Asp Phe Pro580
585 590agt tca gga agt tat cga gta gaa tac aga gtg
gca agt gaa cgt gca 1883Ser Ser Gly Ser Tyr Arg Val Glu Tyr Arg Val
Ala Ser Glu Arg Ala595 600 605gga gga aag
ctg tcc cta gat ttg aat gca ggc tct ata gtg ctt ggc 1931Gly Gly Lys
Leu Ser Leu Asp Leu Asn Ala Gly Ser Ile Val Leu Gly610
615 620 625atg ctg gat att cct tca aca
gga gga ttg cag aag tgg acc acc att 1979Met Leu Asp Ile Pro Ser Thr
Gly Gly Leu Gln Lys Trp Thr Thr Ile630 635
640tct cat ata gtg aat gtg gat tta ggt aca tat aac ttg gga att tat
2027Ser His Ile Val Asn Val Asp Leu Gly Thr Tyr Asn Leu Gly Ile Tyr645
650 655gtt caa aaa gcc agt tgg aat atc aat
tgg att aga att aca aaa gtg 2075Val Gln Lys Ala Ser Trp Asn Ile Asn
Trp Ile Arg Ile Thr Lys Val660 665 670tag
gatacaagag caaaccaatt gtattatttt gaagaaacaa cagctgttga
2128ccataatctt tgttcattga gaatttatcc aactgttata gaatctatca cctttccaga
2188tgtacgcatt gctgatggtt ttgaactaat aaatgaggag attataagtg ctaatgtgtt
2248tgttatatct ttaattttta aaaacaaatt atcaactaac ttttcaattc aggcatggtg
2308tttctctttt taatctgtat ttctaataaa ttaatgtctt taagagttgt tttgtttaca
2368ataaataaag tttgattgtg tgggat
239418673PRTTachypleus tridentatus 18Met Leu Val Leu Leu Cys Cys Val Val
Leu His Val Gly Val Ala Arg1 5 10
15Ile Cys Cys Ser His Glu Pro Lys Trp Gln Leu Val Trp Ser Asp
Glu20 25 30Phe Thr Asn Gly Ile Ser Ser
Asp Trp Glu Phe Glu Met Gly Asn Gly35 40
45Leu Asn Gly Trp Gly Asn Asn Glu Leu Gln Tyr Tyr Arg Arg Glu Asn50
55 60Ala Gln Val Glu Gly Gly Lys Leu Val Ile
Thr Ala Lys Arg Glu Asp65 70 75
80Tyr Asp Gly Phe Lys Tyr Thr Ser Ala Arg Leu Lys Thr Gln Phe
Asp85 90 95Lys Ser Trp Lys Tyr Gly Lys
Ile Glu Ala Lys Met Ala Ile Pro Ser100 105
110Phe Arg Gly Val Trp Val Met Phe Trp Met Ser Gly Asp Asn Thr Asn115
120 125Tyr Val Arg Trp Pro Ser Ser Gly Glu
Ile Asp Phe Ile Glu His Arg130 135 140Asn
Thr Asn Asn Glu Lys Val Arg Gly Thr Ile His Trp Ser Thr Pro145
150 155 160Asp Gly Ala His Ala His
His Asn Arg Glu Ser Asn Thr Asn Gly Ile165 170
175Asp Tyr His Ile Tyr Ser Val Glu Trp Asn Ser Ser Ile Val Lys
Trp180 185 190Phe Val Asn Gly Asn Gln Tyr
Phe Glu Val Lys Ile Gln Gly Gly Val195 200
205Asn Gly Lys Ser Ala Phe Arg Asn Lys Val Phe Val Ile Leu Asn Met210
215 220Ala Ile Gly Gly Asn Trp Pro Gly Phe
Asp Val Ala Asp Glu Ala Phe225 230 235
240Pro Ala Lys Met Tyr Ile Asp Tyr Val Arg Val Tyr Gln Asp
Ala Ser245 250 255Thr Ser Ser Pro Val Gly
Asp Thr Ser Leu Asp Gly Tyr Tyr Phe Val260 265
270Gln Asn Arg His Ser Glu Leu Tyr Leu Asp Val Thr Asp Ala Ser
Asn275 280 285Glu Asp Gly Ala Phe Leu Gln
Gln Trp Ser Tyr Ser Gly Asn Glu Asn290 295
300Gln Gln Phe Asp Phe Glu His Leu Glu Asn Asn Val Tyr Lys Ile Thr305
310 315 320Asn Lys Lys Ser
Gly Lys Ser Leu Asp Val Tyr Asn Phe Gly Thr Glu325 330
335Asn Gly Val Arg Ile Gln Gln Trp Ser Tyr Gly Gly Ala Arg
Asn Gln340 345 350Gln Phe Thr Val Gln Ser
Val Gly Asp Gly Tyr Tyr Lys Ile Ile Pro355 360
365Arg Gly Ser Gly Lys Leu Val Glu Val Ala Asp Phe Ser Lys Asp
Ala370 375 380Gly Gly Lys Ile Gln Gln Trp
Ser Asp Asn Asn Gln Leu Ser Gly Gln385 390
395 400Trp Lys Leu Ile Lys Ser Lys Ser Tyr Ser Lys Leu
Ile Gln Ala Glu405 410 415Ser Tyr Phe Asp
Ser Ser Lys Val Gln Leu Glu Asp Thr Ser Asp Val420 425
430Gly Gly Gly Lys Asn Val Lys Cys Asp Asn Glu Gly Ala Trp
Met Ala435 440 445Tyr Lys Asp Ile Asp Phe
Pro Ser Ser Gly Asn Tyr Arg Ile Glu Tyr450 455
460Arg Val Ala Ser Glu Arg Ala Gly Gly Lys Leu Ser Leu Asp Leu
Asn465 470 475 480Ala Gly
Ser Ile Val Leu Gly Met Leu Asp Val Pro Ser Thr Gly Gly485
490 495Trp Gln Lys Trp Thr Thr Ile Ser His Thr Val Asn
Val Asp Ser Gly500 505 510Thr Tyr Asn Leu
Gly Ile Tyr Val Gln Arg Ala Ser Trp Asn Ile Asn515 520
525Trp Ile Lys Ile Thr Lys Ile Pro Glu Gln Ser Asn Leu Asn
Gln Gly530 535 540Arg Arg Asn Ser Lys Leu
Ile Gln Ala Glu Ser Tyr Phe Ser Tyr Ser545 550
555 560Glu Val Gln Leu Glu Asp Thr Leu Asp Val Gly
Gly Gly Lys Asn Val565 570 575Lys Cys Asp
Lys Glu Gly Ala Trp Met Ala Tyr Lys Asp Ile Asp Phe580
585 590Pro Ser Ser Gly Ser Tyr Arg Val Glu Tyr Arg Val
Ala Ser Glu Arg595 600 605Ala Gly Gly Lys
Leu Ser Leu Asp Leu Asn Ala Gly Ser Ile Val Leu610 615
620Gly Met Leu Asp Ile Pro Ser Thr Gly Gly Leu Gln Lys Trp
Thr Thr625 630 635 640Ile
Ser His Ile Val Asn Val Asp Leu Gly Thr Tyr Asn Leu Gly Ile645
650 655Tyr Val Gln Lys Ala Ser Trp Asn Ile Asn Trp
Ile Arg Ile Thr Lys660 665
670Val191979DNATachypleus tridentatusCDS(101)..(1030) 19gaagacaaga
gagttgaaac aaccatagcc tgtttgctta tgactttcaa taagagatac 60tcggcttaaa
gggaactgac ttattcgtag aggctatacc atg gat atc agt ttc 115Met Asp Ile
Ser Phe1 5ctg gtt ttt atc aca ctg tct atg gct ctc ttc tcg
agc aac gtg aca 163Leu Val Phe Ile Thr Leu Ser Met Ala Leu Phe Ser
Ser Asn Val Thr10 15 20gga acg tca gta
aca tca agg gta cga cgt gga ata aat gaa aaa cat 211Gly Thr Ser Val
Thr Ser Arg Val Arg Arg Gly Ile Asn Glu Lys His25 30
35tgt ggg ttc cga cca gta att aca aga att att ggt gga gga
ata gcg 259Cys Gly Phe Arg Pro Val Ile Thr Arg Ile Ile Gly Gly Gly
Ile Ala40 45 50acg cct cat tca tgg ccg
tgg atg gtt gga att ttc aaa gta aat cct 307Thr Pro His Ser Trp Pro
Trp Met Val Gly Ile Phe Lys Val Asn Pro55 60
65cac cgt ttc ctt tgt ggt gga tct att att aat aaa gtc tct gtt gtt
355His Arg Phe Leu Cys Gly Gly Ser Ile Ile Asn Lys Val Ser Val Val70
75 80 85act gcc gcc cat
tgt ctt gtg acg cag ttt gga aac aga cag aat tat 403Thr Ala Ala His
Cys Leu Val Thr Gln Phe Gly Asn Arg Gln Asn Tyr90 95
100tct atc ttc gta aga gtt gga gcc cat gac ata gac aat tcg
ggt aca 451Ser Ile Phe Val Arg Val Gly Ala His Asp Ile Asp Asn Ser
Gly Thr105 110 115aat tat caa gtg gat aaa
gtt att gtt cac cag ggc tac aaa cac cat 499Asn Tyr Gln Val Asp Lys
Val Ile Val His Gln Gly Tyr Lys His His120 125
130tca cac tac tac gat atc ggt ttg att tta ctc tcg aaa cca gtc gaa
547Ser His Tyr Tyr Asp Ile Gly Leu Ile Leu Leu Ser Lys Pro Val Glu135
140 145tac aac gac aaa ata cag cct gtc tgt
att cct gag ttc aac aaa cct 595Tyr Asn Asp Lys Ile Gln Pro Val Cys
Ile Pro Glu Phe Asn Lys Pro150 155 160
165cac gtg aac ttg aac aat att aag gtc gtc att act ggt tgg
ggt gtt 643His Val Asn Leu Asn Asn Ile Lys Val Val Ile Thr Gly Trp
Gly Val170 175 180act ggg aaa gct act gag
aaa cgt aac gtt ctt cgt gaa ttg gag ttg 691Thr Gly Lys Ala Thr Glu
Lys Arg Asn Val Leu Arg Glu Leu Glu Leu185 190
195ccc gtg gtt aca aac gaa cag tgc aac aaa tct tat cag aca ctc cca
739Pro Val Val Thr Asn Glu Gln Cys Asn Lys Ser Tyr Gln Thr Leu Pro200
205 210ttc tca aaa ttg aac cga gga atc act
aac gac atg att tgt gcg ggg 787Phe Ser Lys Leu Asn Arg Gly Ile Thr
Asn Asp Met Ile Cys Ala Gly215 220 225ttt
ccg gaa gga ggg aaa gat gct tgt cag ggc gac tct ggt ggt ccc 835Phe
Pro Glu Gly Gly Lys Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro230
235 240 245ctg atg tat cag aat cca
aca aca gga aga gtg aaa ata gtt gga gtt 883Leu Met Tyr Gln Asn Pro
Thr Thr Gly Arg Val Lys Ile Val Gly Val250 255
260gta tca ttt ggg ttc gaa tgt gct cgt ccc aac ttc ccc ggt gtt tac
931Val Ser Phe Gly Phe Glu Cys Ala Arg Pro Asn Phe Pro Gly Val Tyr265
270 275acg cgc ctc tcg agc tac gtt aac tgg
ctc cag gaa atc acc ttc gga 979Thr Arg Leu Ser Ser Tyr Val Asn Trp
Leu Gln Glu Ile Thr Phe Gly280 285 290cag
tca ctc gct tct tta ttt gaa gtt gta cca ata ttt ata ccc gag 1027Gln
Ser Leu Ala Ser Leu Phe Glu Val Val Pro Ile Phe Ile Pro Glu295
300 305tga gactgaagat aaatattgaa gagaaatcta
gaataatgta caatataaga 1080agcctgaaat tactgaaata gaaaggcgcg
tgatgagaaa tacgtttcaa attttatttt 1140ttattaactt tattgtgttt aactattctt
tacgtgggac atgaaatata aatctttatt 1200tcttctttat atactttaga ttttcatttc
atctatcttt atcagttttg taatgttact 1260aataatattt cttatggcac ggatcgagcc
tcgtgaatca cagtaaataa taataattat 1320aaaatcacac attattaaaa gcaatagcat
tcagagtgag taacatataa acttcactat 1380gagtggactt ttttattcac attttaagtt
cattactaac tgttgggagg tctttatatt 1440gttgtatatt tatatattaa ttaggttggt
ttagtacatt gttgttaatg gtggaatagg 1500gcgtaggttt taaatgtgtt tgcaaaaaaa
caaacaaaac aagtaatggt ggatgatggt 1560tccaaagtaa ccgaaagaac actttgaaca
tttttataca aaaatttatg ttttaaaata 1620cgagtatata caatcgatct ctaagtacaa
gaaaaactga agtgttcatt caggtttaac 1680agtgcaactt aaatcaacag ttagttgttc
actaaacatt acaatttgat cctttataaa 1740cgctaatact gtttaaacag tcagtaataa
tacagtatca tagcatatca tatatgaagg 1800tattttaaca ttctatatac aaagccagaa
ttgaaaacgg taatattttg tacgattagt 1860gaattattgt ttttaagaac aaactggtat
caaatttaaa atatgaatct gtgatttaat 1920attttttaca acgttctaac ttaccacttt
tgttgtgaat aaaggtgttt acaaatgga 197920309PRTTachypleus tridentatus
20Met Asp Ile Ser Phe Leu Val Phe Ile Thr Leu Ser Met Ala Leu Phe1
5 10 15Ser Ser Asn Val Thr Gly
Thr Ser Val Thr Ser Arg Val Arg Arg Gly20 25
30Ile Asn Glu Lys His Cys Gly Phe Arg Pro Val Ile Thr Arg Ile Ile35
40 45Gly Gly Gly Ile Ala Thr Pro His Ser
Trp Pro Trp Met Val Gly Ile50 55 60Phe
Lys Val Asn Pro His Arg Phe Leu Cys Gly Gly Ser Ile Ile Asn65
70 75 80Lys Val Ser Val Val Thr
Ala Ala His Cys Leu Val Thr Gln Phe Gly85 90
95Asn Arg Gln Asn Tyr Ser Ile Phe Val Arg Val Gly Ala His Asp Ile100
105 110Asp Asn Ser Gly Thr Asn Tyr Gln
Val Asp Lys Val Ile Val His Gln115 120
125Gly Tyr Lys His His Ser His Tyr Tyr Asp Ile Gly Leu Ile Leu Leu130
135 140Ser Lys Pro Val Glu Tyr Asn Asp Lys
Ile Gln Pro Val Cys Ile Pro145 150 155
160Glu Phe Asn Lys Pro His Val Asn Leu Asn Asn Ile Lys Val
Val Ile165 170 175Thr Gly Trp Gly Val Thr
Gly Lys Ala Thr Glu Lys Arg Asn Val Leu180 185
190Arg Glu Leu Glu Leu Pro Val Val Thr Asn Glu Gln Cys Asn Lys
Ser195 200 205Tyr Gln Thr Leu Pro Phe Ser
Lys Leu Asn Arg Gly Ile Thr Asn Asp210 215
220Met Ile Cys Ala Gly Phe Pro Glu Gly Gly Lys Asp Ala Cys Gln Gly225
230 235 240Asp Ser Gly Gly
Pro Leu Met Tyr Gln Asn Pro Thr Thr Gly Arg Val245 250
255Lys Ile Val Gly Val Val Ser Phe Gly Phe Glu Cys Ala Arg
Pro Asn260 265 270Phe Pro Gly Val Tyr Thr
Arg Leu Ser Ser Tyr Val Asn Trp Leu Gln275 280
285Glu Ile Thr Phe Gly Gln Ser Leu Ala Ser Leu Phe Glu Val Val
Pro290 295 300Ile Phe Ile Pro Glu305
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