Novel coronavirus T cell epitope peptide, pMHC and preparation and application of novel coronavirus T cell epitope peptide

A coronavirus, cell antigen technology, applied in the direction of viral peptides, viruses, viruses/phages, etc., can solve the problem that there is no T cell antigen epitope report or suggestion.

Active Publication Date: 2020-08-18
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There are no reports or suggestions on T cell epitopes and their application in immunoassays

Method used

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  • Novel coronavirus T cell epitope peptide, pMHC and preparation and application of novel coronavirus T cell epitope peptide
  • Novel coronavirus T cell epitope peptide, pMHC and preparation and application of novel coronavirus T cell epitope peptide
  • Novel coronavirus T cell epitope peptide, pMHC and preparation and application of novel coronavirus T cell epitope peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Prediction of New Coronavirus HLA-A2 Restricted Epitopes

[0064] 1. Experimental method

[0065] The spike(S) and membrane of 2019-nCoV (MN908947.3), SARS China isolate (DQ182595.1), MERS Riyadh isolate (KF600612.1) and 1997 US common coronavirus isolate (KF530099.1) were taken (M) and nucleocapsid (N) protein sequences, compared with Clustal Omega. Epitopes inconsistent with SARS, MERS and common coronavirus sequences were selected for follow-up research. Simultaneous selection of known HLA-A2 restricted CD8 + T cell epitopes served as positive controls.

[0066] 2. Experimental results

[0067] A large number of high-efficiency and specific T cell antigen epitope candidates were screened.

Embodiment 2

[0068] Example 2 Identification of New Coronavirus HLA-A2 Restricted Epitopes

[0069] 1. Experimental method

[0070] The peptides predicted in Example 1 were artificially synthesized and prepared in different concentrations, namely 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. Take T2-A2 cells in the logarithmic growth state and plant them into 96-well plates, 10 per well 5 , distribute and configure blank wells, negative control peptide (GLQRLGYVL, derived from Zika virus gene code), positive control peptide (influenza A M1 polypeptide, GILGFVFTL) and each synthetic antigen polypeptide, each group has 3 duplicate wells, and the final volume is 200 μL. After incubation at 37°C for 4 hours, centrifuge and wash twice, label with FITC anti-human HLA-A2 (β2m) antibody, incubate at 4°C for 30 minutes in the dark, and detect with flow cytometry. The experiment was carried out 3 times.

[0071] 2. Experimental results

[0072] The result is as figure 1 As shown, the resul...

Embodiment 3

[0075] Example 3 Preparation of pMHC complex of novel coronavirus antigen epitope

[0076] 1. Preparation of HLA-A2 heavy chain and light chain

[0077] 1. Experimental method

[0078] Recombinant construction of genetic engineering technology The heavy chain HLA-A2 (NCBI No. U02935.2) and the light chain β2m (NCBI No. AY187687.1) two target genes were respectively connected to the prokaryotic expression vector pTXB1 through SpeI and NdeI to obtain pTXB1-HLA-A2h and pTXB1-β2m prokaryotic expression plasmids. The plasmid was transformed into BL21 Escherichia coli by heat shock method (42°C for 90 seconds), and sequenced and analyzed. The bacteria liquids successfully identified by sequencing were stored in 30% glycerol at -20°C. Inoculate 10 μL of glycerol bacteria into 4 mL of ampicillin-containing LB medium, culture at 37°C 280 rpm for 16-18 hours, then expand the culture to 400 ml, culture at 37°C 280 rpm for 4 hours, induce expression with 0.1mM IPTG at 24°C 280 rpm for ...

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Abstract

The invention discloses a novel coronavirus T cell epitope peptide, pMHC and the preparation and application thereof. The amino acid sequence of the novel coronavirus T cell epitope peptide is as shown in any one of SEQ ID NO: 1-9. According to the invention, nine novel coronavirus T cell epitope peptides are discovered; the pMHC compound monomer is prepared by using the pMHC compound; the pMHC complex polymer can be used for detecting antigen-specific T cells in peripheral blood of a novel coronavirus infected rehabilitative person and used for an in-vitro T cell activation experiment, and the novel coronavirus T cell epitope peptides can be applied to immunodetection related to novel coronaviruses.

Description

technical field [0001] The present invention relates to the field of biotechnology, more specifically, relates to a novel coronavirus T cell antigen epitope peptide, pMHC and its preparation and application. Background technique [0002] The new coronavirus is the pathogen of a new type of infectious pneumonia that occurred at the end of 2019. It has caused a pandemic in more than 160 countries and regions around the world, with more than 15,000 deaths. The source of the virus is not yet clear, but gene sequencing shows that it is highly homologous to bat SARS-like coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV). There is no specific drug report yet. The immune response mechanism of the human immune system to the new coronavirus, including antigen recognition, immune clearing mechanism, and immune memory protection mechanism, is not clear. [0003] According to the research on SARS and other...

Claims

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Application Information

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IPC IPC(8): C07K14/165C07K14/74G01N33/569
CPCC07K14/005C07K14/70539C12N2770/20022G01N33/56972G01N33/56983
Inventor 陈国兵邱聪龄王鹏程罗钧洪高利娟
Owner JINAN UNIVERSITY
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