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SARS-CoV-2 specific polypeptides and application thereof

A sars-cov-2, specific technology, applied in the direction of specific peptides, peptides, peptide sources, etc., can solve problems such as evaluating the level of T cell immunity

Pending Publication Date: 2021-12-28
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the use of tetramer technology to evaluate the T cell immunity level of SARS-CoV-2 infection recovered population or vaccinated population.

Method used

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  • SARS-CoV-2 specific polypeptides and application thereof
  • SARS-CoV-2 specific polypeptides and application thereof
  • SARS-CoV-2 specific polypeptides and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Screening of SARS-CoV-2 specific polypeptides

[0030] Use the peptide prediction software to predict the M and N proteins of SARS-CoV-2 to obtain peptides of 15-18 amino acids. After dissolving the peptides with DMSO, the peptides of the M and N proteins were mixed into a peptide library. The PBMCs of convalescents were cultured with the peptide library as a stimulus, and the cells were harvested after 9 days of culture.

[0031] The synthesized peptides are numbered and arranged in a matrix, horizontal N groups, vertical N groups, horizontal groups and vertical groups are crossed, and the polypeptides of each horizontal group and vertical group are mixed into a secondary peptide library. The situation of IFN-γ produced by the secondary peptide library was detected by Elispot technology, and 2 functional peptides were obtained by crossing the horizontal and vertical secondary peptide libraries that produced IFN-γ (such as the amino acid sequence in Table 1 su...

Embodiment 2

[0034] Example 2 Preparation of Polypeptide-MHC Tetramer

[0035] (1) Preparation of polypeptide / MHC complex:

[0036] 1) Add the amino acid sequence GGGLNDIFEAQKIEWHE capable of linking biotin to the C-terminus of the HLA-A*1101 (Genbank accession number MT462157.1) gene to construct the recombinant plasmid pET28a-HLA-A*1101-Bio, and insert the B2m gene (Genbank accession number is AAA39668.1) was connected with the vector to construct the recombinant vector pET28a-B2m, and the plasmids were transformed into Escherichia coli BL21 to obtain recombinant bacteria E.coli / pET28a-B2m and E.coli / pET28a-HLA-A*1101-Bio ;

[0037] 2) Cultivate the Escherichia coli BL21 carrying the plasmid in step 1) at 37°C and add 1mmol / L IPTG to induce protein expression, collect the bacteria, ultrasonically disrupt the bacteria, and dissolve the precipitate after high-speed centrifugation (12000rpm, 10min) In the dissolution buffer (6mol / L guanidine hydrochloride, 10% glycerol, 50mmol / L Tris pH8....

Embodiment 3

[0053] Example 3 Application of Polypeptide / MHC Tetramer in T Cell Analysis

[0054] Using the characteristics of high affinity and high specificity of SARS-CoV-2 specific peptide / MHC tetramer to detect SARS-CoV-2 infection and population T cells after vaccine immunization, evaluate SARS-CoV-2 infection, recovery The effect of cellular immunity, isolation and cloning of T cells, etc. in patients and post-vaccination populations, etc., the following steps were used to evaluate the level of T cell immunity in patients who recovered from SARS-CoV-2 infection:

[0055] 1) Select PBMCs from patients who recovered from SARS-CoV-2 infection with HLA-A*1101 type, and use the synthetic peptide library as a stimulus to stimulate and cultivate PBMCs for 9 days;

[0056] 2) Harvest the cultured cells and wash them twice with FACS buffer / staining buffer (PBS+0.5% BSA);

[0057] 3) Staining of cell surface molecules. Add antibodies (such as FITC-CD8, APC-CD4, PerCp-CD3, PE-Tetramer).

[...

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Abstract

The invention discloses SARS-CoV-2 specific polypeptides and application thereof, and belongs to the field of immunodetection. The invention provides two specific polypeptides aiming at SARS-CoV-2; a polypeptide-MHC tetramer is prepared from the corresponding polypeptides; the polypeptide-MHC tetramer is used for detecting T cells of SARS-CoV-2 infected convalescent patients; the positive rates are 3.63% and 2.37% respectively and are obviously higher than 0.061% and 0.096% of a negative control group; the affinity of the polypeptide-MHC and TCR on the surfaces of specific T cells is effectively increased; the technology can be used as an effective tool for T cell evaluation, can also be used for separating and cloning the specific T cells, is combined with a single cell sequencing technology to separate specific TCR, and can be used as a T cell activation reagent and the like; and the technology has a relatively high application value in the aspect of researching the T cells of people infected with the SARS-CoV-2 or vaccinated with the SARS-CoV-2.

Description

technical field [0001] The invention relates to a SARS-CoV-2 specific polypeptide and an application thereof, belonging to the field of immune detection. Background technique [0002] Since the novel coronavirus SARS-CoV-2 was first discovered in December 2019, it has spread rapidly around the world and has been declared a serious public health emergency of international concern (PHEIC) by the World Health Organization. A year and a half into the current SARS-CoV-2 pandemic, which has resulted in more than 213 million cases of coronavirus disease 2019 (COVID-19) and 4.4 million deaths, remains a serious challenge to global public health. The development of safe and effective vaccines is one of the important means to control the spread of SARS-CoV-2. T cells play a very important role in virus clearance. In the evaluation of the immune effect of SARS-CoV-2 vaccine, the evaluation of T cell immune response is particularly important. Evaluation of T cell immunity in patients ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C07K19/00A61K39/215A61P31/14G01N33/569
CPCC07K14/005C07K14/70539C07K19/00A61K39/12A61P31/14G01N33/56966C12N2770/20022C12N2770/20034Y02A50/30
Inventor 刘军张杰高福
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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