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Mycobacterium tuberculosis specific CD4+T cell epitope peptide P12 and application thereof

A technology of mycobacterium tuberculosis, epitope peptide, applied in the field of immunology

Inactive Publication Date: 2016-04-13
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently known tuberculosis epitope peptides are mainly identified in European and American populations, most of which are HLA-A*0201-restricted, but for African and Southeast Asian populations where tuberculosis is most prevalent, there is no suitable epitope peptide to use

Method used

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  • Mycobacterium tuberculosis specific CD4+T cell epitope peptide P12 and application thereof
  • Mycobacterium tuberculosis specific CD4+T cell epitope peptide P12 and application thereof
  • Mycobacterium tuberculosis specific CD4+T cell epitope peptide P12 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Obtaining of HLA-DRB1*09:01 Restricted Epitope Peptides

[0022] The present invention obtains the frequency distribution information of each genotype of the HLA-DRB1 locus in the Chinese population from the HLA gene frequency database AFND (AlleleFrequencyNetDatabase, AFND), wherein the HLA-DRB1 gene frequency is about 15%, which is the highest frequency on the HLA-DRB1 locus single genotype. Prediction of HLA-DRB1*09:01-restricted CD4 contained in antigenic protein using bioinformatics algorithm + T cell epitope.

[0023] The present invention comprehensively utilizes various technical means of bioinformatics and immunoinformatics to detect HLA-DRB1*09:01-restricted CD4 contained in the antigenic protein sequence of Mycobacterium tuberculosis. + T epitope peptides are predicted and analyzed, and through a large number of screenings, epitope peptides with high affinity to HLA molecules are screened out and identified by immunological experiments. The sc...

Embodiment 2

[0026] Example 2: ELISA assay detects epitope peptide P12 induces T cells to secrete IFN-γ

[0027] The experimental method is as follows:

[0028] Peripheral blood mononuclear cells (PBMC) were isolated from tuberculosis patients with HLA-DRB1*09:01 genotype by density gradient method. Dilute 20ml of collected peripheral blood with PBS, slowly add it to the top of the lymphocyte separation medium, the volume ratio of the two is 2:1, and then centrifuge at 800g / min for 30min. After centrifugation, the liquid is divided into three layers. Between the first layer and the second layer, a white film is formed. Use a dropper to carefully draw PBMC into a new centrifuge tube, add more than 5 times the volume of PBS to wash, centrifuge at 800g / min for 10min, repeat the wash once, and use the freezing solution ( 90% FBS+10% DMSO) frozen cells, stored in liquid nitrogen.

[0029] ELISA experiment: Resuscitate PBMC from patients with HLA-DRB1*09:01 genotype at 37°C, 5% CO 2 Stand o...

Embodiment 3

[0032] Example 3: ELISpot assay to detect the frequency of antigen-specific T cells secreting IFN-γ induced by epitope peptide P12

[0033] The experimental method is as follows:

[0034]Peripheral blood mononuclear cells (PBMC) from tuberculosis patients were separated by density gradient method. Dilute 20ml of collected peripheral blood with PBS, slowly add it to the top of the lymphocyte separation medium, the volume ratio of the two is 2:1, and then centrifuge at 800g / min for 30min. After centrifugation, the liquid is divided into three layers. Between the first layer and the second layer, a white film is formed. Use a dropper to carefully draw PBMC into a new centrifuge tube, add more than 5 times the volume of PBS to wash, centrifuge at 800g / min for 10min, repeat the wash once, and use the freezing solution ( 90% FBS+10% DMSO) frozen cells, stored in liquid nitrogen.

[0035] ELISpot experiment: resuscitating PBMCs of HLA-DRB1*09:01 and non-HLA-DRB1*09:01 tuberculosi...

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Abstract

The present invention discloses a Mycobacterium tuberculosis specific CD4+T cell epitope peptide P12 and application thereof. The present invention comprehensively utilizes bioinformatics and immunoinformatics technologies, screens epitope peptide with anti-TB activity by using the antigen of Mycobacterium tuberculosis; the screened epitope peptide P12 and HLA-DRB1*09: 01 molecules have high affinity; and the epitope peptide can be widely identified by CD4+T cells of patients with TB. Since HLA-DRB1*09:01 genotype is widely carried by Chinese people, the Mycobacterium tuberculosis specific HLA-DRB1*09:01 restricted Th1 epitope peptide is not found currently. The determination of epitope peptide P12 provides a theoretical basis and a novel solution for the development of TB vaccines, diagnostic reagents and therapeutic drugs, and is of great significance for the prevention and treatment of tuberculosis.

Description

technical field [0001] The invention belongs to the technical field of immunology, in particular to Mycobacterium tuberculosis-specific CD4 + T cell epitope peptide and its application. Background technique [0002] Tuberculosis is an infectious disease induced by Mycobacterium tuberculosis infection with the highest infection rate in the world, and its case fatality rate is second only to AIDS. In 2012 alone, there were 8.6 million new cases of tuberculosis worldwide and 1.3 million deaths. The new features of the tuberculosis epidemic are: the emergence and spread of drug-resistant tuberculosis cases; the co-infection of tuberculosis and AIDS is intensifying. For these two types of patients, there are currently no effective preventive measures. China is one of the countries with the heaviest burden of tuberculosis in the world, and it is time to develop tuberculosis vaccines and therapeutic drugs suitable for the Chinese population. [0003] Mycobacterium tuberculosis ...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10A61K39/04A61P31/06G01N33/68G01N33/569
Inventor 马骊刘苏东
Owner SOUTHERN MEDICAL UNIVERSITY
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