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SARS-CoV virus structural protein infusion protein and its high yield expression and purification and uses

A technology of structural protein and fusion protein, applied in the direction of positive-sense single-stranded RNA virus, virus, viral peptide, etc., can solve problems such as hidden dangers of SARS vaccine safety

Inactive Publication Date: 2006-12-27
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The third is the safety of the vaccine. The research on the pathogenic mechanism of SARS-CoV virus is limited. How SARS-CoV virus causes acute lung injury, heart failure, and the way of immune system collapse is not clear. The preparation of SARS vaccine in the prior art safety hazards in
And prior art fails to solve this problem, therefore, further invention and research are needed to prepare a safe and effective SARS-CoV virus vaccine

Method used

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  • SARS-CoV virus structural protein infusion protein and its high yield expression and purification and uses
  • SARS-CoV virus structural protein infusion protein and its high yield expression and purification and uses
  • SARS-CoV virus structural protein infusion protein and its high yield expression and purification and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0136] Example 1 Whole-gene artificial synthesis of SEQ ID No: 1

[0137] The whole gene of SEQ ID No: 1 was artificially synthesized by entrusting Shanghai (China) Boya Biotechnology Co., Ltd. with a gene synthesis method known in the prior art.

[0138] The known whole-gene artificial synthesis method uses 100-base oligonucleotide primers corresponding to each other and corresponding PCR amplification primers. The two parts of the oligonucleotide primers overlap by 20 bases to form a complete gene. After connection, Annealing, PCR amplification, and synthesis of the whole gene.

Embodiment 2

[0139] Example 2 Plasmid construction and identification

[0140] (1) Obtaining of PCR products:

[0141] A primer design and synthesis

[0142]S317: forword: 5'GGCGCTAGCCAGCGACCTGGACCGCTGC3'

[0143] reverse: 5'CGCGGATCCGTCGGGGAAGCGCACGACGTC3'

[0144] S510: forword: 5'GGCGCTAGCCAGCGACCTGGACCGCTGC3'

[0145] reverse: 5'CGCGGATCCGTCACGGTGGCGGGGGCGTTC3'

[0146] S685: forword: 5'GGCGCTAGCCAGCGACCTGGACCGCTGC3'

[0147] reverse: 5'CGCGGATCCGTGGCGCCCAGGCTCATGGTG3'

[0148] S900: forword: 5'GGCGCTAGCCAGCGACCTGGACCGCTGC3'

[0149] reverse: 5'CGCGGATCCGTCTCGTACAGCACGTTCTG3'

[0150] S1148: forword: 5'GGCGCTAGCCAGCGACCTGGACCGCTGC3'

[0151] reverse: 5'CGCGGATCCGTCAGGTCCACGTCGGGGCTG3'

[0152] S1190: forword: 5'GGCGCTAGCCAGCGACCTGGACCGCTGC3'

[0153] Reverse: 5'CTCACATGTATGGATCCTTCTGCTCGTACTTGCCCAG3'

[0154] S318-510: forword: 5'GGCGCTAGCCATCACCAACCTGTGCCCC3'

[0155] reverse: 5'CGCGGATCCGTCACGGTGGCGGGGGCGTTC3'

[0156] S318-1190: forword: 5'GGCGCTAGCCATCACCAACCTGTGCCCC3'...

Embodiment 3

[0218] Example 3 Establishing a constant expression cell line

[0219] (1) Restriction endonuclease AvrII (purchased from NEW ENGLAND BioLabs  Inc, USA) digested about 10 μg of the recombinant plasmid, took a small amount of the digested product and electrophoresed to confirm that the digested product was complete, purified the remaining digested product with a purification kit (purchased from V-Gene Company), recovered the DNA, and removed it. enzymes and proteins etc.

[0220] (2) Digest the cells with trypsin, add medium, and blow into single cells. Count 2 x 10 5 cells into each well of a 6-well cell culture plate.

[0221] (3) After 24 hours, use liposome (lipofectamine TM 2000 purchased from Invitrogen TM ) transfection water (negative control) and 0.5 μg digested and purified DNA (operate according to the operation method provided by the kit).

[0222] (4) After 48 hours, it was divided into 12-well cell culture plates (the cells in the wells of each 6-well cell...

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Abstract

The invention relates to fusion protein of SARS-Cov virus structural protein and its high expression and purification in mammalian cell and its usage. The construction of said fusion protein is X-Y-z, and X is S or M or E or N selected from SARS-CoV virus construction protein, or their arbitrary short form; Y is the connection part with 0-20 amino acid; Z is Fc and its modification or other protein tag. The invention also provides the method of expressing and purifying said fusion protein in mammalian cell for the batch preparation or industrial production. The fusion protein can be used to prepare genetic engineering vaccine preventing SARS-CoV virus infection, solvent box for checking SARS-CoV virus, and to sift drug anti SARS-CoV virus infection with S protein combined with its acceptor ACE2. The invention can detect combination of S protein of SARS-CoV virus with ACE2, which reduces ACE2 expression, and result in or exacerbate acute lung damage, then it modifies combined segment, which can improve safety of preventing SARS-CoV virus vaccine.

Description

technical field [0001] The present invention relates to the fusion protein of SARS-CoV viral structural protein and its high-level expression in mammalian cells; The application of said fusion protein in the preparation of genetic engineering vaccines and medicines for preventing and treating SARS-CoV virus infection; and the use of said fusion protein Use of the fusion protein in the preparation of a kit for detecting SARS-CoV virus infection. The present invention also relates to the discovery of the toxic fragment of SARS-CoV virus structural protein S; and various vaccines designed to prevent SARS-CoV virus infection. technical background [0002] The pathogen of Severe Acute Respiratory Syndrome (SARS) is a new type of coronavirus (SARS-CoV), which has a wide range of hosts and fast transmission speed, and can be transmitted through droplets or even air, which is extremely harmful. Therefore, research on the prevention, treatment and detection of SARS-CoV virus infecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K16/00C12N15/62C12N15/00C12N15/63C12N5/08A61K39/215G01N33/569G01N33/68
CPCC12N2770/20034C07K2319/00C07K14/005A61K39/215C12N2770/20022A61K2039/55566A61K39/12A61P11/00A61P31/14
Inventor 蒋澄宇郭峰饶栓关冰环奕杨鹏
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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