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Specific T cell epitope peptide P38 screened from novel coronavirus S protein whole proteome and application thereof

A coronavirus, specific technology, applied in the direction of viral peptides, applications, viruses, etc., to achieve the effect of good immune stimulation

Pending Publication Date: 2021-12-03
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But not validated on T cells from COVID-19 patients
[0008] Moreover, there is no report on the specific T cell epitope peptide obtained by screening the entire proteome of the new coronavirus S protein

Method used

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  • Specific T cell epitope peptide P38 screened from novel coronavirus S protein whole proteome and application thereof
  • Specific T cell epitope peptide P38 screened from novel coronavirus S protein whole proteome and application thereof
  • Specific T cell epitope peptide P38 screened from novel coronavirus S protein whole proteome and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Collection of blood samples from COVID-19 convalescent patients and unexposed population

[0034] Blood samples were collected from discharged patients with COVID-19 within two weeks after discharge, and PBMCs were immediately isolated and stored in liquid nitrogen. All COVID-19 patients are required to meet the discharge criteria for COVID-19 patients in the Diagnosis and Treatment Program for Novel Coronavirus Infected Pneumonia (7th Edition) promulgated by the General Office of the Chinese Health Commission. Blood PBMC samples from unexposed persons (no SARS-CoV-2 infection, no new crown vaccine) were collected as negative controls.

Embodiment 2

[0035] Example 2 SARS-CoV-2 Spike protein peptide library

[0036] According to the amino acid sequence of the SARS-CoV-2 S protein (access number: YP_009724390.1), the length of the peptide was designed to be 15-mers, and two consecutive peptides overlapped by 5 amino acids. Finally, a total of 127 peptides covering the full length of the S protein were obtained (P1-P127), the amino acid sequence of the polypeptide is shown in Table 1. The 127 peptides were arranged in order into a checkerboard list of 12(C1-C12)×11(R1-R11), see Table 2, and 23 peptide groups were obtained.

[0037] Table 1 Peptide sequences of Spike protein peptide library

[0038]

[0039]

[0040] Table 2 Peptide checkerboard list

[0041] Peptide group C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 R1 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 R2 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23 P24 R3 P25 P26 P27 P28 P29 P30 P31 P32 P33 P34 P35 ...

Embodiment 3

[0042] Example 3 Detection of the proportion of T cells secreting the cytokine IFN-γ

[0043] 1×10 6 Each PBMC was incubated with the peptide group to be tested (the final concentration of each peptide was 2.5 μg / mL) or an equal volume of culture medium (non-stimulation group) for 20 hours (37°C, 5% CO 2 ), Golgi blocker was added 5 hours before the end of incubation. After the end of the culture, the dead cells, cell surface markers (CD3, CD4, CD69) and intracellular cytokines (IFN-γ) were stained according to the reagent instructions, and detected by flow cytometry (BD FACSCantoTMⅡ), and 15- 200,000 lymphocytes were analyzed using the software Flowjo 10, see flow gating strategy figure 1 .

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Abstract

The invention provides a specific T cell epitope peptide P38 screened from a novel coronavirus S protein whole proteome and application thereof. The amino acid sequence of the specific T cell epitope peptide P38 is shown as SEQ No. P38. The screened T cell epitope peptide is subjected to specificity verification, so that cross reaction in healthy people is eliminated, and the T cell epitope peptide can be used as a specific antigen for detecting new coronary patients. The screened specific T cell epitope peptide P38 is detected in more peripheral blood mononuclear cell samples of patients in a novel coronavirus pneumonia recovery period, and the positive reaction rates of the specific T cell epitope peptide P38 in CD4+T and CD8+T cells are 48.0 percent and 24.0 percent respectively. The invention also provides an HLA allele type capable of identifying the epitope peptide. The epitope peptide has potential to be used as one of antigen components of a new coronary vaccine, and can be used for developing a detection kit.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a specific T cell epitope peptide P38 for novel coronavirus spike (spike, S) protein whole proteome screening and applications thereof. Background technique [0002] In antiviral immunity, neutralizing antibodies can prevent virus invasion. At the same time, the role of T cell immune response in clearing viruses cannot be ignored. CD4 + T cells help induce memory B cell maturation and antibody responses; CD8 + T cells assist in virus clearance, and memory CD8 + T cells can provide long-term protection and effectively prevent secondary infections. Severe acute respiratory syndrome coronavirus (severe acute respiratory syndrome corona virus, SARS-CoV) and SARS-CoV-2 belong to the same genus of betacoronavirus, the clinical symptoms of the disease caused by the two, disease outcome, and the spread of the virus itself The rules all have certain similarities. Long-term mo...

Claims

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Application Information

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IPC IPC(8): C07K14/165C12N15/50G01N33/569A61K39/215A61P31/14
CPCC07K14/005G01N33/56983G01N33/56972A61K39/12A61P31/14C12N2770/20022C12N2770/20034A61K2039/5154A61K2039/53G01N2333/165
Inventor 王晓辉杨峥嵘冯铁建邹璇何建凡
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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