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Recombinant baculovirus and use thereof

A recombinant baculovirus, encoding technology, applied in the direction of virus, virus/phage, double-stranded DNA virus, etc., can solve the problem of vitamin K-dependent blood coagulation factors not being carboxylated by γ-glutamyl

Active Publication Date: 2014-10-01
SYSMEX CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, it is known that vitamin K-dependent blood coagulation factors obtained by using an expression system using mammalian cells are not carboxylated by γ-glutamyl at all

Method used

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  • Recombinant baculovirus and use thereof
  • Recombinant baculovirus and use thereof
  • Recombinant baculovirus and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Production and expression of recombinant baculovirus

[0046] (1) Subcloning of genes encoding GGCX, NQO1 and VKOR respectively

[0047] Based on the reported base sequence of the human GGCX gene (NCBI Acc.No.EU847509), the base sequence of the human NQO1 gene (NCBI Acc.No.AK312368) and the base sequence of the human VKOR gene (NCBI Acc.No.AY521634 ), primer sets designed for subcloning each gene. The base sequence of each primer is shown below. In addition, the nucleotide sequence of an appropriate restriction endonuclease site is added to each primer.

[0048] (i) Primer set for GGCX gene

[0049] F: 5'-GGGGTACCATGGCGGTGTCTGCCGGGTCCGC-3' (SEQ ID NO: 6)

[0050] R: 5'-GCTCTAGAGAACTCTGAGTGGACAGGATCA-3' (SEQ ID NO: 7)

[0051] (ii) Primer set for NQO1 gene

[0052] F: 5'-GAAGATCTATGGTCGGCAGAAGAGCACTGATCGTA-3' (SEQ ID NO: 8)

[0053] R: 5'-GCTCTAGATTTTCTAGCTTTGATCTGGTTGTCAGTT-3' (SEQ ID NO: 9)

[0054] (iii) Primer set for VKOR gene

[0055] F: 5'-GGGGTA...

Embodiment 2

[0066] Example 2: Expression of vitamin K-dependent proteins in silkworms and exploration of γ-glutamylglutamyl carboxylation

[0067] (1) Subcloning the genes encoding factor X and prothrombin respectively

[0068] Based on the base sequence of the human factor X gene (hereinafter also referred to as hFX gene) (NCBI Acc.No.BC_040125) and the base sequence of human prothrombin gene (hereinafter also referred to as hPTH gene) published in the database (NCBI Acc.No.NM_000506), designed primer sets for cloning each gene. The sequence of each primer is shown below. In addition, the nucleotide sequence of an appropriate restriction endonuclease site is added to each primer.

[0069] (i) Primer set for hFX gene

[0070] F: 5'-AAGGTACCCGGGGATCCATGGGGCGCCCACTG-3' (SEQ ID NO: 12)

[0071] R: 5'-AATCTAGATCACTTTAATGGAGAGGACGTTAT-3' (SEQ ID NO: 13)

[0072] (ii) Primer set for hPTH gene

[0073] F: 5'-AAGAATTCATGGCCAACACCTTCTTGGAGGAG-3' (SEQ ID NO: 14)

[0074] R: 5'-AATCTAGACTACTT...

Embodiment 3

[0100] Example 3: Discussion on conditions for γ-glutamyl carboxylation of vitamin K-dependent proteins in silkworms

[0101] The FX single-expression virus produced in the above-mentioned Example 2 and each expression virus produced in the above-mentioned Example 1 were mixed until the virus titer reached the following ratio, and they were inoculated into silkworm larvae (species: Jinqiujinhe. Silkworm eggs were purchased from Ueda Silkworm Co., Ltd., and artificially reared to larvae by Sysmex Co., Ltd. (test area 1-4). In addition, as a control, only the virus expressing FX alone was inoculated into silkworm larvae (experimental area 5).

[0102] Seven days after virus inoculation, body fluids were extracted from infected larvae. To confirm the expression level of Factor X, a portion of the obtained body fluid was analyzed by SDS-PAGE and Western blot using an anti-Factor X antibody (Enzyme Research Laboratories). In addition, in order to detect factor X of γ-glutamyl car...

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Abstract

The present invention provides a recombinant baculovirus. The baculovirus has a genome into which a gene encoding ³-glutamyl carboxylase (GGCX) and a gene encoding DT-diaphorase (NQO1) are incorporated. The present invention further provides a method for producing a recombinant vitamin K-dependent protein by using the recombinant baculovirus.

Description

technical field [0001] The present invention relates to a recombinant baculovirus and a kit for producing a recombinant vitamin K-dependent protein containing the virus. In addition, the present invention also relates to host cells infected with recombinant baculoviruses. Furthermore, the present invention also relates to a method for producing a recombinant vitamin K-dependent protein. Background technique [0002] Vitamin K is a cofactor for γ-glutamyl carboxylase (GGCX), responsible for the post-translational modification of various vitamin K-dependent proteins. In the presence of vitamin K, GGCX carboxylates designated glutamic acid residues of various vitamin K-dependent proteins to γ-carboxyglutamic acid (Gla). This γ-glutamyl carboxylation is known to be extremely important for the biological functions of vitamin K-dependent proteins (for example, blood coagulation, bone metabolism, signal transmission, etc.). For example, blood coagulation factor II (prothrombin) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N5/10C12R1/93
CPCC12N9/6429A01K2207/05A01K2267/01C12N9/6432C12N9/6437C12P21/02C12N9/0036C12N9/6464C12N9/644C12Y401/0109C12Y106/05002C12N9/88C12N2710/14143C07K14/745C12N15/86A01K2227/706Y02P20/52
Inventor 坂东孝彦菅井睦美
Owner SYSMEX CORP