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EP-3517962-B1 - AN AGENT FOR MEASURING ENDOTOXIN

EP3517962B1EP 3517962 B1EP3517962 B1EP 3517962B1EP-3517962-B1

Inventors

  • MIZUMURA, HIKARU
  • AIZAWA, MAKI
  • ODA, TOSHIO

Dates

Publication Date
20260506
Application Date
20120228

Claims (9)

  1. A method for producing an endotoxin-measuring agent comprising factors which are a recombinant factor C of horseshoe crab, a recombinant factor B of horseshoe crab, and a recombinant pro-clotting enzyme of horseshoe crab; wherein the factor C is a recombinant protein obtained by being expressed using insect cells as a host, and does not have any peptide sequence attached at the C-terminus, wherein the method comprises the steps (A) to (D) below: (A) a step of incorporating each of DNAs (1) to (3) into a viral DNA; (1) a DNA encoding the factor C derived from Tachypleus tridentatus, (2) a DNA encoding the factor B, (3) a DNA encoding the proclotting enzyme; (B) a step of infecting insect cells with the virus into which said each DNA was incorporated; (C) a step of allowing the insect cells infected with said each virus to express the factors encoded by said each DNA; and (D) a step of eliminating said virus from each of the expressed factors, wherein the virus is a baculovirus.
  2. A method for producing an endotoxin-measuring agent comprising a recombinant factor C of horseshoe crab, a recombinant factor B of horseshoe crab, and a recombinant pro-clotting enzyme of horseshoe crab; wherein the factor C is a recombinant protein obtained by being expressed using insect cells stably expressing the factor C, and does not have any peptide attached at the C-terminus, wherein the method comprises the steps (A) to (C) below: (A) a step of incorporating each of DNAs (1) to (3) into a vector; (1) a DNA encoding the factor C derived from Tachypleus tridentatus, (2) a DNA encoding the factor B, (3) a DNA encoding the pro-clotting enzyme; (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 DNA was incorporated, to express the protein encoded by said each DNA.
  3. The method according to claim 1, wherein the baculovirus is a nucleopolyhedrovirus (NPV).
  4. The method according to claim 3, wherein the nucleopolyhedrovirus is Autographa californica NPV or Bombix mori NPV.
  5. The method according to any one of claims 1 to 4, wherein said insect cell is selected from the group consisting of Sf9, Sf21, SF+, and High-Five.
  6. The method according to any one of claims 1 to 5, wherein a cultivation of the insect cells is carried out at 27°C to 28°C with shaking.
  7. The method according to any one of claims 1 to 6, wherein the amino acid sequence of the factor C encoded by the DNA comprises an amino acid sequence having an identity of not less than 80% to the full length of the amino acid sequence shown in SEQ ID NO:2 and having factor C activity, wherein the factor C activity is an activity by which factor C becomes active in the presence of endotoxin, to activate factor B.
  8. The method according to any one of claims 1 to 6, wherein the amino acid sequence of the factor C encoded by the DNA comprises an amino acid sequence having an identity of not less than 90% to the full length of the amino acid sequence shown in SEQ ID NO:2 and having factor C activity, wherein the factor C activity is an activity by which factor C becomes active in the presence of endotoxin, to activate factor B.
  9. The method according to any one of claims 1 to 6, wherein the amino acid sequence of the factor C encoded by the DNA comprises an amino acid sequence shown in SEQ ID NO:2.

Description

Field of invention The present invention relates to a method for producing an endotoxin-measuring agent. Background to the Invention Endotoxin is a lipopolysaccharide existing on the outer membrane of the cell wall of Gram-negative bacteria, and known to be a strong pyrogen. Further, it is known that even a small amount of endotoxin causes various disease states due to bacterial infection, such as release of inflammatory cytokines due to macrophage activation and induction of endotoxin shock, in addition to fever. Therefore, detection of endotoxin in pharmaceuticals such as those for injection; water; medical equipments; and the like is important. Further, endotoxin is considered to be the main cause of shock in Gram-negative bacterial infection, and hence, the presence or absence of infection and/or a pharmaceutical effect can be judged by measuring endotoxin in the blood. Further, it is known that infection of American horseshoe crab (Limulus polyphemus) with Gram-negative bacteria causes intravascular coagulation, and this phenomenon has been used for detection of endotoxin. That is, a method for measuring endotoxin using a blood cell extract of a horseshoe crab (horseshoe crab amebocyte lysate; hereinafter also referred to as "lysate") is known (e.g., Non-patent Document 1). This method is called "limulus test", and uses a cascade reaction of various proteins existing in the lysate, which reaction is caused by contacting of endotoxin with the lysate. A schematic diagram of the cascade reaction is shown in Fig. 1. Upon contacting of endotoxin with the lysate, factor C existing in the lysate is activated to produce active-type factor C. This active-type factor C activates factor B existing in the lysate, to produce active-type factor B. This active-type factor B then activates a proclotting enzyme existing in the lysate, to produce a clotting enzyme. This clotting enzyme hydrolyzes a specific portion in the coagulogen molecule existing in the lysate. By this, coagulin gel is produced, to cause coagulation of the lysate. Thus, by measuring the coagulation reaction of the lysate, endotoxin can be measured. Further, also by allowing a clotting enzyme to react with a synthetic substrate to cause color reaction, endotoxin can be measured. For example, a clotting enzyme reacts with a synthetic substrate t-butoxycarbonyl-leucyl-glycyl-arginyl-pNA (Boc-Leu-Gly-Arg-pNA) to hydrolyze its amide bond, and thereby pNA is released. Thus, by preliminarily including the synthetic substrate in the reaction system, endotoxin can be quantified by measurement of the absorbance (405 nm) of the coloring substance (pNA). Further, it is known that the cascade reaction system can be reconstructed using factor C, factor B, and a proclotting enzyme, which were purified from lysate of a Japanese horseshoe crab (Non-patent Document 2). Further, a case wherein a recombinant factor C derived from a Southeast Asian horseshoe crab Carcinoscorpius rotundicauda; and a recombinant factor B and a recombinant proclotting enzyme derived from a Japanese horseshoe crab Tachypleus tridentatus; were used to reconstruct the cascade reaction system is known (Patent Document 1). Further, a system for detecting endotoxin by using a recombinant factor C derived from a Southeast Asian horseshoe crab Carcinoscorpius rotundicauda and a substrate that reacts with active-type factor C to release a fluorescent substance is known (Patent Document 2). This system is commercially available as an endotoxin detection system (commercial name: PyroGene (registered trademark); Lonza). However, in order to use the lysate, or the naturally occurring factor C, factor B, and proclotting enzyme prepared therefrom, it is necessary to capture horseshoe crabs and collect blood therefrom. Hence, in view of conservation of biological resources or the like, it is difficult to supply these components unlimitedly. Therefore, a technique to easily and rapidly produce a reagent for detection of endotoxin at a low cost has been demanded. A method of preparing Factor C purified from horseshoe crabs, is described in Patent Document 3. Patent Document 4 describes producing factor C by a viral method using Sf9 cells. Further, Non-patent Document 3 describes production of horseshoe crab factor C in a yeast expression system, and Non-patent Document 4 describes a factor C isolated from a horseshoe crab hemolymph. Further, in cases where a recombinant factor C, recombinant factor B, and recombinant proclotting enzyme are used, any of the above-described cases requires 1 hour or more for the measurement, and a detection sensitivity in the order of 0.001 EU/mL has not been achieved. Therefore, a technique to rapidly and highly sensitively measure endotoxin has been demanded. Prior Art Documents Patent Documents [Patent Document 1] WO 2008/004674[Patent Document 2] US 6,849,426 B[Patent Document 3] EP 0291856 A[Patent Document 4] WO 03/002976 A2 Non-patent Documents [Non-patent Document 1] I