Agent for measuring endotoxin

Abstract

A method for rapidly and highly sensitively measuring endotoxin is provided. Endotoxin is measured using an endotoxin-measuring agent comprising the proteins (1) to (3) below, each of which is a recombinant protein obtainable by being expressed using insect cells as a host: (1) a factor C derived from Tachypleus tridentatus , which factor C does not have a His-tag sequence at the C-terminus; (2) a factor B of a horseshoe crab; and (3) a proclotting enzyme of a horseshoe crab.

1 . An endotoxin-measuring agent comprising the proteins (1) to (3) below, each of which is a recombinant protein obtainable by being expressed using insect cells as a host:
 (1) a factor C derived from  Tachypleus tridentatus , which factor C does not have His-tag sequence at the C-terminus;   (2) a factor B of a horseshoe crab; and   (3) a proclotting enzyme of a horseshoe crab.   
     
     
         2 . The measuring agent according to  claim 1 , wherein said factor B and said proclotting enzyme are derived from  Tachypleus tridentatus.    
     
     
         3 . The measuring agent according to  claim 1 , wherein said factor C is the protein (A) or (B) below; said factor B is the protein (C) or (D) below; and said proclotting enzyme is the protein (E) or (F) below:
 (A) a protein comprising the amino acid sequence shown in SEQ ID NO:2;   (B) a protein comprising the amino acid sequence shown in SEQ ID NO:2 but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, which protein has factor C activity;   (C) a protein comprising the amino acid sequence shown in SEQ ID NO:4;   (D) a protein comprising the amino acid sequence shown in SEQ ID NO:4 but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, which protein has factor B activity;   (E) a protein comprising the amino acid sequence shown in SEQ ID NO:6;   (F) a protein comprising the amino acid sequence shown in SEQ ID NO:6 but which includes substitution, deletion, insertion, or addition of one or several amino acid residues, which protein has proclotting enzyme activity.   
     
     
         4 . A method for producing the measuring agent according to  claim 1 , the method comprising the steps (A) to (C) below:
 (A) a step of incorporating each of the DNAs (1) to (3) below into a viral DNA:
 (1) a DNA encoding a factor C derived from  Tachypleus tridentatus , which factor C does not have His-tag sequence at the C-terminus; 
 (2) a DNA encoding a factor B of a horseshoe crab; and 
 (3) a DNA encoding a proclotting enzyme of a horseshoe crab; 
   (B) a step of infecting insect cells with the virus into which said each DNA was incorporated; and   (C) a step of allowing the insect cells infected with said each virus to express the protein encoded by said each DNA.   
     
     
         5 . A method for producing the measuring agent according to  claim 1 , the method comprising the steps (A) to (C) below:
 (A) a step of incorporating each of the DNAs (1) to (3) below into a vector:
 (1) a DNA encoding a factor C derived from  Tachypleus tridentatus , which factor C does not have His-tag sequence at the C-terminus; 
 (2) a DNA encoding a factor B of a horseshoe crab; and 
 (3) a DNA encoding a proclotting enzyme of a horseshoe crab; 
   (B) a step of introducing the vector, into which said each DNA was incorporated, into insect cells to incorporate said each DNA into the chromosome of the insect cells; and   (C) a step of allowing the insect cells, into which said each DNA was incorporated, to express the protein encoded by said each DNA.   
     
     
         6 . The method according to  claim 4 , wherein said DNA encoding factor C is the DNA (A) or (B) below; said DNA encoding factor B is the DNA (C) or (D) below; and said DNA encoding proclotting enzyme is the DNA (E) or (F) below:
 (A) a DNA comprising the nucleotide sequence shown in SEQ ID NO:1;   (B) a DNA which hybridizes with the complementary sequence of the full length or a part of the nucleotide sequence shown in SEQ ID NO:1 under stringent conditions, and encodes a protein having factor C activity;   (C) a DNA comprising the nucleotide sequence shown in SEQ ID NO:3 or 8;   (D) a DNA which hybridizes with the complementary sequence of the full length or a part of the nucleotide sequence shown in SEQ ID NO:3 or 8 under stringent conditions, and encodes a protein having factor B activity;   (E) a DNA comprising the nucleotide sequence shown in SEQ ID NO:5 or 9;   (F) a DNA which hybridizes with the complementary sequence of the full length or a part of the nucleotide sequence shown in SEQ ID NO:5 or 9 under stringent conditions, and encodes a protein having proclotting enzyme activity.   
     
     
         7 . A method for measuring endotoxin in a test sample, the method comprising a step of mixing the measuring agent according to  claim 1  with the test sample and a step of measuring progress of cascade reaction. 
     
     
         8 . The method according to  claim 7 , which comprises a step of adding a substrate for detection of progress of cascade reaction to a reaction system. 
     
     
         9 . The method according to  claim 8 , which further comprises a step of calculating the endotoxin level in the test sample on the basis of reaction of said substrate.