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New coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen, specific neutralizing antibody thereof and application

A polypeptide antigen, sars-cov-2 technology, applied in the direction of viral peptides, viruses/phages, antibodies, etc., can solve problems such as the lack of broad-spectrum vaccines and rapid detection methods for detecting anti-S2 antibodies.

Active Publication Date: 2021-09-07
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there is currently no broad-spectrum vaccine against SARS-CoV-2 S2 and a rapid test for detecting anti-S2 antibodies

Method used

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  • New coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen, specific neutralizing antibody thereof and application
  • New coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen, specific neutralizing antibody thereof and application
  • New coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen, specific neutralizing antibody thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] A new coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen determination and antigen-specific detection method

[0058] Using biological software to analyze the amino acid sequence of the S2 protein of SARS-CoV-2, from the hydrophilicity, antigenic index, surface site index and the conserved possible antigenic fragment of the S2 protein, the amino acid sequence is DPLQPELDSFKEELDKYFKNHTSPDVDLGDIS (SEQ ID NO: 1) The polypeptide. After comparison, the peptide shown in SEQ ID NO: 1 has no change in the S protein of SARS, SARS-CoV-2 of the β-coronavirus genus, and all variants of SARS-CoV-2 found so far.

Embodiment 2

[0060] 1. Preparation of ELISA detection kit using polypeptide antigen

[0061] The polypeptide antigen preparation kit described in Example 1 is composed as follows:

[0062] 1. ELISA plates coated with polypeptide antigens;

[0063] 2. Enzyme-labeled antibody: horseradish peroxidase-labeled mouse anti-human IgG;

[0064] 3. Substrate solution preparation: 100mmol / L citric acid solution (21g citric acid dissolved in deionized water, dilute to 1L) 24.3mL, 200mmol / L Na 2 HPO 4 12H 2 O (71.6g Na 2 HPO 4 12H 2 O (dissolved in deionized water, dilute to 1L) 25.7mL and mix well, add 50mg of tetramethylbenzidine (TMB), add 50μL of 30% H 2 o 2 .

[0065] 4. Stop solution 2M H 2 SO 4 : Take 89 mL of distilled water and concentrated H 2 SO 4 11 mL to mix.

[0066] 5. Washing solution: add 0.5mL Tween-20 to 1000mL 10mmo1 / L PBS with pH 7.4;

[0067] 6. Negative control (human negative serum) and positive control (human SARS-CoV-2 inactivated positive serum).

[0068] 2. Pre...

Embodiment 3

[0078] A kind of preparation method of SARS-CoV-2 triple broad-spectrum polypeptide tandem fusion protein

[0079] 1) Design and amplify the primers containing the pColdⅠ linearization vector and 3 corresponding gene fragments containing SEQ ID NO: 1: the upstream primer for amplifying the pColdⅠ linearization vector is located at positions 337-361 of the pColdⅠ plasmid; the downstream primer for amplifying the pColdⅠ linearization vector is located at Positions 295-318 of the pColdI plasmid. The amplified fragment gene containing SEQ ID NO: 1 is located at position 3415-3510 of the SARS-CoV-2S gene; the upstream primer for amplifying the first fragment has 15 reverse complementary primers downstream of the pColdⅠ linearized vector at the 5' end Base, the downstream then adds the reverse complementary sequence with the corresponding base of expressing Linker GGGGS; The upstream and downstream primers of the amplification second fragment add the corresponding base sequence and ...

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Abstract

The invention provides a new coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen, a specific neutralizing antibody thereof and application, and belongs to the technical field of virus immunodetection. The new coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen has an amino acid sequence shown as SEQ ID NO: 1, and can be specifically combined with a new coronavirus antibody through reaction with SARS-CoV-2 human positive serum. Based on the polypeptide sequence, triple SARS-CoV-2 broad-spectrum polypeptide tandem fusion protein is prepared by utilizing PCR, prokaryotic expression and protein purification technologies, a trimer mode of SARS-CoV-2S protein in a natural state is simulated, the fusion protein is used as an antigen to immunize mice, the SARS-CoV-2 specific neutralizing antibody can be generated, and the neutralizing antibody has good application prospects in SARS-CoV-2 anti-infection treatment, vaccine research and development and detection kit development.

Description

technical field [0001] The invention belongs to the technical field of virus immune detection, and in particular relates to a novel coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen and its specific neutralizing antibody and application. Background technique [0002] The novel coronavirus SARS-CoV-2 is a highly contagious virus. The S protein of SARS-CoV-2 contains two subunits: S1 and S2. The S1 subunit binds to the cellular receptor angiotensin-converting enzyme 2 (ACE2) through its RBD, while the S2 subunit mediates the fusion of the virus with the cell membrane through the fusion peptide (FP). Due to its essential role in the fusion process during infection, S2 could serve as a target for broad protection. At present, the frequent emergence of escape mutants of SARS-CoV-2 has brought challenges to the current vaccine strategy for the new crown vaccine with immunodominant receptor binding domain (RBD). [0003] However, there is currently no broad-spectrum vacc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C07K19/00C12N15/50C12N15/70C07K16/10G01N33/569A61K39/42A61P31/14
CPCC07K14/005C07K16/10C12N15/70G01N33/56983A61P31/14C07K2319/00C12N2770/20022G01N2333/165A61K2039/505C07K14/165G01N33/6854C12N15/10
Inventor 叶建强李拓凡阚秋琪秦爱建万志敏邵红霞谢泉
Owner YANGZHOU UNIV
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