Method for producing recombinant prothrombin, vector DNA, and reagent kit

Patent metadata

Field	Value
Publication number	US-9447402-B2
Application number	US-201414202046-A
Country	US
Kind code	B2
Filing date	Mar 10, 2014
Priority date	Mar 15, 2013
Publication date	Sep 20, 2016
Grant date	Sep 20, 2016

Abstract

The present invention provides a method for producing recombinant prothrombin. The method comprises: providing a vector DNA into which a gene encoding a tag and a gene encoding prothrombin are incorporated, wherein the tag is selected from the group consisting of MBP, SUMO, and NusA; and expressing a tag fusion type prothrombin in a lepidopteran insect or cultured cells of the lepidopteran insect.

First claim

What is claimed is:

1. A method for producing recombinant prothrombin, comprising:
providing a vector DNA into which a gene encoding a tag and a gene encoding prothrombin are incorporated, wherein the tag is selected from the group consisting of MBP, SEQ ID NO: 2, and NusA; and
expressing a tag fusion type prothrombin in a lepidopteran insect or cultured cells of the lepidopteran insect.

2. The method according to claim 1 , further comprising:
obtaining a soluble fraction containing the tag fusion type prothrombin from the lepidopteran insect or the cultured cells of the lepidopteran insect after the expressing step.

3. The method according to claim 1 , wherein the gene encoding a tag is incorporated into the vector DNA so that the tag is fused to the N terminal of prothrombin.

4. The method according to claim 1 , wherein a gene encoding a polypeptide having the amino acid sequence set forth in any one of SEQ ID NOS: 7 to 9 is incorporated into the vector DNA.

5. The method according to claim 1 , wherein the lepidopteran insect is Bombyx mori.

6. The method according to claim 1 , wherein the expressing step comprises infecting the lepidopteran insect or the cultured cells of the lepidopteran insect with a baculovirus comprising the vector DNA.

7. The method according to claim 1 , wherein a gene encoding a protein secretory signal sequence is incorporated into the vector DNA.

8. The method according to claim 7 , wherein the protein secretory signal sequence is at least one selected from the group consisting of a prothrombin-derived secretory signal sequence, a Bombyx mori -derived 30K signal sequence, and a Bombyx mori -derived SP signal sequence.

Assignees

• Sysmex Corp

Inventors

• Bando Takahiko
• Sugai Mutsumi

Classifications

• C12N9/6429 Primary

  Thrombin (3.4.21.5) · CPC title

• C07K2319/20

  containing a tag with affinity for a non-protein ligand · CPC title

• C12N2710/14143

  viral genome or elements thereof as genetic vector · CPC title

• C07K2319/35

  containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin · CPC title

• C12N9/647

  Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups · CPC title