SARS coronavirus polypeptide antigen and application thereof

A coronavirus and polypeptide antigen technology, applied to the SARS coronavirus polypeptide antigen and its application field, can solve the problems of no antigen immunized animals, inconvenient operation, and high detection cost, saving detection time, reducing detection cost, and improving detection. The effect of accuracy

Inactive Publication Date: 2007-12-12
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, there is no report of using its whole protein or partial polypeptide sequence as an antigen to immunize animals
[0003] Currently commonly used SARS clinical detection methods

Method used

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  • SARS coronavirus polypeptide antigen and application thereof
  • SARS coronavirus polypeptide antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation of specific antibody against 3a protein

[0043]Using biological software, the antigenicity of the 3a protein was analyzed (the virus strain is SARS coronavirusGZ50; Viruses; ssRNA positive-strand viruses, no DNA stage; Nidovirales; Coronaviridae; Coronavirus; Group 2 species. GeneBank number: AAS00004. The source is the University of Hong Kong Professor Zheng Bojian), selected three of the proteins (amino acid sequences with high antigenicity, and named them LH21 (4-24 Amino acids SEQ ID NO: 1FRMRFTLGSITAQPVKIDNAS), LH22 (211-231 Amino acids SEQ ID NO: 2YYQLESTQITTDTGIENATFF) and LH23 (251-271 Amino acids SEQID NO: 3SSGVANPAMDPIYDEPTTTTS). These three polypeptides were synthesized by chemical methods (Gill Biochemical (Shanghai) Co., Ltd.). The polypeptides were dissolved in PBS buffer respectively, and passed through glutaraldehyde and carrier protein BSA Coupling. After 2 hours of chemical coupling, add glysin to terminate the reaction, and dialyze the re...

Embodiment 2

[0047] Detection of 3a protein expression in virus-infected cells using anti-LH21 antibody

[0048] Get the FRhK-4 cells infected by the above-mentioned SARS-CoV, and wash the cells several times with PBS buffer. Directly use 1×SDS cell lysis buffer and lyse the cells on ice for 15 minutes. The cell lysate was ultrasonically pulverized, boiled in water for 5 minutes, used for SDS polyacrylamide gel electrophoresis, and incubated with anti-LH21 polyclonal antibody and monoclonal antibody at 37 degrees Celsius for 2 hours, stained to observe the results of western blot. (figure 1)

Embodiment 3

[0050] Application of epitope polypeptide LH-21, that is, ELISA method to detect anti-SARS coronavirus antibody in samples

[0051] The epitope polypeptide was coated at a concentration of 5 ng / mL; it was co-incubated with a series of concentration gradient SARS coronavirus antibody controls and samples to be tested at 37 degrees Celsius for one hour; Incubate the sample at 37°C for two hours; add HRP-labeled anti-human antibody as a secondary antibody and incubate at 37°C for one hour; add enzyme reaction substrate after washing, and read OD at 450nm; the antigen can be obtained after comparing with the standard curve concentration. (figure 2)

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Abstract

The invention relates to a kind of polypeptide for SARS coronavirus and its application. The amnio acid squence is SEQ ID NO: 1 or its derivant squence. The sifted antigen epitope LH21 is N section of protein coded by ORF3a gene, its immunity is strong, and the clinical diagnosis value is good. The antibody prepared with said epitope can be used to check virus antigen.

Description

Background technique [0001] Atypical pneumonia (SARS) is caused by a newly discovered coronavirus (severe acute respiratory syndrome associated coronavirus, Nidovirales; Coronaviridae; Coronavirus; Group 2 species). The viral genome consists of a single-stranded RNA containing 14 open reading frames (ORFs). The currently known proteins encoded by viruses include: lipid bilayer membrane and its rod-shaped membrane protein S, envelope protein E, membrane glycoprotein M, and membrane inner capsid protein N, as well as some proteases and replicases, but most Whether the ORFs of ARS encode proteins, and their functions are unclear (Rota PA, Oberste MS, The genome sequence of the ARS-associated coronavirus. Science. 2003 May 30; 300(5624): 1399-404). [0002] ORF3a is one of the SARS virus genes that has been studied more recently. ORF3a (for example: the sequence is SEQ ID NO: 4) encodes a new viral protein with a full length of 274 amino acids (for example: the sequence is SEQ I...

Claims

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Application Information

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IPC IPC(8): C07K14/165C07K16/18G01N33/543G01N33/577
Inventor 孙兵郑伯健吕伟徐可
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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