Pro-clotting enzyme, and method for detection of endotoxin or (1-3)-beta-d-glucan using the same

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.

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; and   detecting 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; and   detecting 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; and   detecting 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; and   detecting 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→3)-β-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→3)-β-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→3)-β-D-glucan; and   detecting (1→3)-β-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→3)-β-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→3)-β-D-glucan comprises factor G derived from a horseshoe crab and/or recombinant factor G. 
     
     
         22 . A method of detecting (1→3)-β-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→3)-β-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→3)-β-D-glucan; and   detecting (1→3)-β-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→3)-β-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→3)-β-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→3)-β-D-glucan; and   detecting (1→3)-β-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→3)-β-D-glucan.   
     
     
         24 . The (1→3)-β-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→3)-β-D-glucan comprises factor G derived from a horseshoe crab and/or recombinant factor G. 
     
     
         25 . A (1→3)-β-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→3)-β-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→3)-β-D-glucan; and   detecting (1→3)-β-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→3)-β-D-glucan,   wherein the kit contains, as cascade reaction proteins, exclusively a pro-clotting enzyme according to  claim 11  and recombinant factor G.