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Bat SARS-like coronavirus spike protein immunity determining area and preparation method and application thereof

A coronavirus and protein technology, which is applied to the immune-determining region of the bat SARS-like coronavirus spike protein and the fields of its preparation and use, can solve the problems of inability to neutralize human SARS coronavirus and the like

Inactive Publication Date: 2012-09-26
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The applicant's previous work found that the bat serum infected by bat SARS-like coronavirus had cross-reaction with human SARS-CoV S protein, but could not neutralize human SARS-CoV, indicating that the difference between bat SARS-like coronavirus and human SARS-CoV S protein There is a big difference between

Method used

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  • Bat SARS-like coronavirus spike protein immunity determining area and preparation method and application thereof
  • Bat SARS-like coronavirus spike protein immunity determining area and preparation method and application thereof
  • Bat SARS-like coronavirus spike protein immunity determining area and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0052] Embodiment 1: design and amplify bat SARS-like coronavirus S gene truncated fragment, its steps are:

[0053] Based on the sequence of the bat SARS-like coronavirus S gene (GenBank sequence number: DQ071615, position in the genome nucleotide sequence: 21486-25211) obtained in the previous work of our laboratory, primers were designed for PCR amplification to obtain Rp3-S1 and Its five truncated S target genes. The primers are as follows:

[0054] 1-666: Forward: 5'-GGCCGAATTCATGAAAATTTTAAT-3',

[0055] Reverse: 5'-CAGTGTCGACTTAAGACATAGTGTAAGCCAC-3';

[0056] 1-81aa: Forward: 5’-GGCCGGATCCATGAAAATTTTAAT-3’,

[0057] Reverse: 5'-ACTTGTCGACGTAGGTAAACCTAT-3';

[0058] 82-280aa: Forward: 5'-GGCCGGATCCTTTGACAATCCTAT-3';

[0059] Reverse: 5'-CTCAGTCGACAATAGCATTGGTAA-3';

[0060] 280-455aa: Forward: 5'-TGCCGGATCCATTGATTGTGCTCA-3',

[0061] Reverse: 5'-CAGTGTCGACTTAAGAAAGGTCTCTCTCAAAA-3';

[0062] 429-574aa: forward: 5'-GGACGGATCCACTGCAAAGCAGGATCAA-3',

[0063] Reverse:...

Embodiment 2

[0068] The construction of bat SARS-like coronavirus S truncated protein recombinant expression vector and its expression and purification, its steps are:

[0069] The recovered PCR product and expression vector pET32a plasmid were digested with restriction endonucleases BamHI and XhoI (products of Takara Company). Enzyme digestion reaction: 1 μl each of BamHI and XhoI, 2 μl of 10-fold H buffer, 50-100 ng of PCR product or pET32a plasmid, and sterile water to a total volume of 20 μl. After 1 hour at 37°C, the digested product was recovered by DNA, and then the PCR product was ligated with the expression vector pET32a with T4 DNA ligase (product of Takara Company). Ligation reaction: T4 DNA ligase (1U / μl) l μl, the molar ratio of PCR product to expression vector pET32a is 3:1, and the total amount of DNA is 0.1 μg, 5 times ligase reaction buffer 4 μl, add sterile water to The total volume was 20 μl and left at 16°C for 24 hours.

[0070] Add 2 μl of the ligation reaction solu...

Embodiment 3

[0074] ELISA reaction of bat SARS-like coronavirus S truncated fragment and S DNA immunized mouse serum.

[0075] The anti-full-length S serum of mice comes from the following plasmid immunized with DNA: Rp3-S constructed in pCDNA3.1 (+) codon-optimized (Ren et al, 2008, Journal of Virology, Difference in Receptor Usage between Severe Acute Respiratory Syndrome (SARS) Coronavirus and SARS-Like Coronavirus of Bat Origin)) and pCDNA3.1(+) empty vector plasmid (Introgen) were used as negative controls. 30 μg of the plasmid was diluted with 30 μl of PBS and injected into 6-8 week-old female BALB / c mice by electric shock in vivo. The experiment was divided into 2 groups (see zhou et al, 2009, BBRC, Immunogenicity difference between the SARS coronavirus us and the batSARS-like coronavirus spike(S)proteins), with 5 mice in each group. The mice were immunized at 0, 3 and 5 weeks respectively, and the mouse serum was collected at the eighth week. The sera of 5 mice immunized with Rp3...

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Abstract

The invention discloses a bat SARS-like coronavirus spike protein immunity determining area and a preparation method and application thereof. The method comprises the following steps of: A, amplifying by taking the bat SARS-like coronavirus S gene as a template design primer; B, performing enzyme digestion of the fragment obtained by the amplification by use of BamHI and XhoI, connecting to an expression vector pET32a, and sequencing to assure correctness; and C, purifying the recombinant plasmid, transforming the BL21 competent cell, culturing the monoclonal antibody, and performing 30-degree induction in a culture medium with the final concentration of 0.3mMIPTG; collecting the thalli, performing ultrasonic breaking, and then purifying with a HisTag purification kit; and after the purification, detecting the concentration of protein in SDS-PAGE to obtain the target protein. The method is simple and easy to implement, convenient to operate and easy in production; the peptide has the best immunogenicity; and a method of applying the protein to the identification of mice S monoclonal antibody epitope has high specificity, and is simple to operate and easy to repeat.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a bat SARS-like coronavirus spike protein (Rp3-S) immunodetermining region, and also relates to a bat SARS-like coronavirus spike protein (Rp3-S) immunodetermining region The preparation method also relates to the use of a bat SARS-like coronavirus spike protein (Rp3-S) immunodetermining region. Background technique [0002] SARS coronavirus is the causative agent of SARS that broke out in China in 2002-2003. The bat SARS-like coronavirus has a high homology with the SARS coronavirus. Since it was first discovered by our research group in 2005, there have been reports on similar viruses around the world, but there are not many reports on the immunogenicity of bat SARS-like coronaviruses. The University of Hong Kong believes through informatics analysis that the virus has the risk of escaping and spreading to humans. Studies have shown that the main difference betwe...

Claims

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Application Information

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IPC IPC(8): C07K14/165C12N15/50C12N15/63G01N33/68
Inventor 周鹏韩正刚石正丽
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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