Method for measuring endotoxin
AssigneeSeikagaku Corporation
InventorsHikaru Mizumura, Maki Aizawa, Toshio Oda
Life of this patent
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Abstract
A method for rapidly and highly sensitively measuring endotoxin relies on an endotoxin-measuring agent, which includes a factor C derived from Tachypleus tridentatus that does not have His-tag sequence at the C-terminus, a factor B of a horseshoe crab, and a proclotting enzyme of a horseshoe crab. Each of these proteins can be a recombinant protein obtainable by being expressed using a stably expressing cell line of an insect cell as a host.
Claims8
1-3. (canceled)
A method for producing an endotoxin measuring agent, comprising:
- (A) incorporating each of the DNAs (1) to (3) below into three viral DNAs respectively:
- (1) a DNA encoding a factor C, which DNA is DNA (A′) or (B′) below, which factor C does not have His-tag sequence or any peptide attached at the C-terminus:
- (A′) a DNA encoding a protein consisting of an amino acid sequence shown in SEQ ID NO:2;
- (B′) a DNA encoding a protein having a sequence consisting of an amino acid sequence having an identity of 90% or higher to the amino acid sequence shown in SEQ ID NO:2, wherein the protein has factor C activity greater than the activity of natural factor C under the same condition;
- (2) a DNA encoding a factor B of a horseshoe crab;
- and
- (3) a DNA encoding a proclotting enzyme of a horseshoe crab;
- (B) infecting insect cells with the three viruses, into which said each DNA was incorporated;
- (C) allowing the insect cells infected with said each virus to express the protein encoded by said each DNA;
- (D) eliminating said virus from the expressed protein;
- and
- (E) formulating the endotoxin measuring agent comprising the proteins from which the virus was eliminated.
5-9. (canceled)
A method for producing a factor C recombinant protein for use in an endotoxin measuring agent, comprising:
- (a) incorporating a DNA encoding the factor C recombinant protein into a virus, wherein the factor C recombinant protein encoded by the DNA is a protein (A′) or (B′) below, and does not have His-tag sequence or any peptide attached at the C-terminus:
- (A′) a protein consisting of an amino acid sequence shown in SEQ ID NO:2;
- (B′) a protein consisting of an amino acid sequence having an identity of 90% or higher to the amino acid sequence shown in SEQ ID NO:2, wherein the protein has factor C activity greater than the activity of natural factor C under the same condition;
- (b) infecting insect cells with the virus, into which said DNA was incorporated;
- (c) allowing the insect cells infected with said virus to express the protein encoded by said DNA;
- and
- (d) eliminating said virus from the expressed protein.
Dependencies are inferred from the claim text.