Method for expressing and producing recombinant human blood coagulation factors VII in animal cells

A technology for coagulation factors and cells, applied in the field of genetic engineering, can solve the problems of lack of modification and processing, high production cost and low expression level.

Active Publication Date: 2010-12-08
SUZHOU ZELGEN BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] For example, Escherichia coli can express a variety of proteins as a host, but its existence (1) lacks post-translational modification and processing of eukaryotic proteins, such as cleavage, glycosylation, formation of disulfide bonds, etc.; (2) the expressed protein Insoluble inclusion bodies are often formed, and complex renaturation is required to restore conformation and activity; (3) Bacterial proteins are not conducive to purification and other disadvantages
[0010] The Saccharomyces cerevisiae system also has limitations: (1) the yield is

Method used

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  • Method for expressing and producing recombinant human blood coagulation factors VII in animal cells
  • Method for expressing and producing recombinant human blood coagulation factors VII in animal cells
  • Method for expressing and producing recombinant human blood coagulation factors VII in animal cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Construction of pSA vector and pSA / FVII vector

[0082] see figure 1 , first use the conventional PCR method to obtain the FVII gene and the elements shown in the following table:

[0083] element

Features

pCMV (CMV promoter)

transcription initiation

pT7 (T7 promoter)

In vitro mRNA sequencing initiation

MCS (multicloning restriction site)

for cloning the gene of interest

BGH PolyA (Bovine Growth Factor polyA site)

Transcription termination and post-transcriptional RNA processing signals

SV40 early promoter

Initiates expression of Neo(R) selectable marker genes

Neo(R) gene

Selectable marker genes for cell line selection

SV40polyA site

Transcription termination and post-transcriptional RNA processing signals

[0084] The pCMV (CMV promoter), pT7 (T7 promoter), MCS (multicloning restriction site), BGHPolyA (bovine growth factor polyA site), SV40 early promoter, Neo(R) gene, and...

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PUM

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Abstract

The invention provides a method for expressing and producing recombinant human blood coagulation factors VII in animal cells, in particular an expression vector for expressing exogenous protein in the animal cells. The expression vector contains an expression cassette for expressing the exogenous protein, wherein the expression cassette comprises the following elements from 5' to 3': (a) a first promoter; (b) a polyclone locus and an optional coding sequence of the exogenous protein; (c) a first polyA sequence; (d) a second promoter; (e) a selected marker gene; and (f) a second polyA signal sequence. The expression vector is particularly suitable for expressing the FVII efficiently in the animal cells.

Description

technical field [0001] The invention relates to the field of genetic engineering. More specifically, the present invention relates to a method for expressing and producing recombinant human blood coagulation factor VII in animal cells. The present invention also provides an expression vector particularly suitable for expressing FVII highly in animal cells. Background technique [0002] Coagulation factor VII (FVII) is a vitamin K-dependent zymogen with serine proteolytic activity, which is mainly synthesized in the liver. Most of FVII in plasma is a single-chain inactive zymogen with a concentration of 0.5 ug / ml (10 nmol / l). There are traces of activated FVIIa in plasma, at a concentration of 10-110 pmol / l. [0003] After vascular damage, tissue factor (TF) exposure, FVII or activated FVII (FVIIa) can form a complex with TF, and under the action of FXa, thrombin, etc., the arginine 152 and isoleucine in the FVII chain The 153 position is cleaved into double strands and a...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12P21/02
Inventor 盛泽林
Owner SUZHOU ZELGEN BIOPHARML
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