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Application of mycobacterium tuberculosis T cell epitope protein Rv1566c-444

A technology of rv1566c-444 and Mycobacterium tuberculosis, applied in the fields of molecular biology and immunology, can solve the problem of not being able to cause immune protection response, and achieve the effects of low cost, reducing false negatives, and improving detection sensitivity

Pending Publication Date: 2022-06-28
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Because the immunogenicity of the protein antigens contained in the commercially available tuberculosis diagnostic reagents and the existing research-stage anti-tuberculosis vaccines still cannot elicit the required sufficient immune protective response, the search for antigens with good immunogenicity for tuberculosis The development of diagnostic reagents and new vaccines is crucial

Method used

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  • Application of mycobacterium tuberculosis T cell epitope protein Rv1566c-444
  • Application of mycobacterium tuberculosis T cell epitope protein Rv1566c-444
  • Application of mycobacterium tuberculosis T cell epitope protein Rv1566c-444

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1 T cell epitope prediction and selection of T cell epitope-enriched sequences

[0041] The sequence of the Mycobacterium tuberculosis Rv1566c gene was obtained from the US National Center for Biotechnology Information (NCBI), see SEQ ID No. 1, and the corresponding protein sequence, see SEQ ID No. 2. T-cell epitope prediction software TEpredict and IEDB-AR were used to predict the Rv1566c gene that can bind to human HLA-A02 supertype alleles (including HLA-A *0201, *0202, *0203, *0206 motifs) T-cell epitopes, a total of 29 human T-cell epitopes were predicted in the Rv1566c gene, which can be divided into six epitope-concentrated regions. The present invention designs a sequence contained in the Rv1566c gene that retains all 29 T cell epitopes, and the targeting DNA sequence is named Rv1566c-444n, see SEQ ID No. 3, located at the 109-552 nucleotide position of the Rv1566c gene , the corresponding protein is defined as Rv1566c-444, see SEQ ID No.4. The distrib...

Embodiment 2

[0042] Example 2 Validation of T cell epitopes

[0043] The gene sequences of the six epitope concentration regions of Rv1566c were sent to Shanghai Sangon Biotechnology Co., Ltd. to synthesize the corresponding six epitope peptides, and about 20 ml of peripheral blood from pulmonary tuberculosis patients were collected, and peripheral blood lymphocytes were separated. The peptides stimulated peripheral blood lymphocytes from 10 pulmonary tuberculosis patients respectively, and the ELISPOT test was carried out using the Mycobacterium tuberculosis effector T cell detection kit (enzyme-linked immunospot method) purchased from Beijing Jinhao Pharmaceutical according to the instructions. If the ELISPOT result of a pulmonary tuberculosis patient is positive according to the kit standard, this epitope polypeptide is determined to be a positive human T cell epitope polypeptide. The sixth polypeptide among the six epitope polypeptides of Rv1566c ( figure 1 D6) was identified as a pos...

Embodiment 3

[0048] Example 3 Gene amplification, expression and purification of recombinant proteins pET-32a-Rv1566c and pET-32a-Rv1566c-444

[0049] Using the genomic DNA of Mycobacterium tuberculosis H37Rv as a template, the complete sequence of the Rv1566c gene and the epitope sequence Rv1566c-444n were amplified by PCR technology. After digestion with EcoRI and hind, it was ligated into pET-32a plasmid and transformed into Escherichia coli BL21(DE3). Protein expression was induced using 1 mM isopropyl β-d-thiogalactoside (IPTG) in the medium. After culturing at 37°C for 3.5 hours, the cells were collected by centrifugation at 4°C (4000 rpm, 10 min, 4°C). Expression levels and forms of Rv1566c and Rv1566c-444 in E. coli were detected by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by denaturation of the inclusion body proteins with 8M urea and nickel - Nitrilotriacetic acid (Ni-NTA) chromatography to purify the protein. Then, a low-concentration...

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Abstract

The invention relates to the technical field of molecular biology and immunology, and particularly discloses application of a T cell epitope protein Rv1566c-444 of mycobacterium tuberculosis. The invention provides an application of a mycobacterium tuberculosis T cell epitope protein Rv1566c-444 in preparation of a tuberculosis detection reagent and a vaccine. Wherein the amino acid sequence of the mycobacterium tuberculosis T cell epitope protein Rv1566c-444 is shown as SEQ ID NO.4, or is an amino acid sequence which is formed by replacing, deleting or adding one or more amino acids to the sequence and has the same antigenicity and immunogenicity. The mycobacterium tuberculosis T cell epitope protein Rv1566c-444 provided by the invention can reduce false negative caused by T cell stimulation by an intrinsic protein antigen of a kit, so that the detection sensitivity is improved. The cell immune response is good, and the vaccine can be widely applied to the related fields of auxiliary diagnosis of tuberculosis, epidemiological monitoring, infection screening and the like, and provides a new thought for the development of a new anti-tuberculosis vaccine.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and immunology, in particular to the application of a Mycobacterium tuberculosis T cell epitope protein Rv1566c-444. Background technique [0002] Tuberculosis is a zoonotic infectious disease caused by Mycobacterium tuberculosis. Its morbidity, prevalence and mortality are high, and it is prone to drug resistance and multidrug resistance, and drug resistance is increasing. Rapid point-of-care diagnostic tests, new vaccines or effective preventive treatments are priorities to end the TB epidemic. [0003] The early diagnosis of tuberculosis is crucial for tuberculosis control, and the lack of rapid, sensitive and specific diagnostic tools poses a huge challenge to tuberculosis control. The protective immune mechanism against tuberculosis is still not fully understood, and cellular immunity, especially the T lymphocyte immune response in the circulation and infection sites, is believed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/35C12N15/31G01N33/569G01N33/577G01N33/58G01N33/543A61K39/04A61P31/06
CPCC07K14/35G01N33/5695G01N33/577G01N33/581G01N33/54393A61K39/04A61P31/06G01N2469/10
Inventor 万康林刘海灿范雪亭李马超
Owner ICDC CHINA CDC
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