Mycobacterium tuberculosis antigen protein Rv0585c and application of T cell epitope peptide thereof
A technology of Mycobacterium tuberculosis, rv0585c, applied in the fields of molecular biology and immunology, can solve the problems of poor sensitivity, poor specificity of tuberculosis, high price, etc., and achieve the effects of facilitating quality control, reducing false positives, and improving sensitivity
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Embodiment 1
[0044] Example 1 Cloning of Mycobacterium tuberculosis antigen gene Rv0585c and protein expression and purification
[0045] Rv0585c (GI: 15607725) is a conserved membrane protein encoded by the Mycobacterium tuberculosis H37Rv genome, containing 795 amino acids, and its amino acid sequence is shown in SEQ ID NO:4. According to its coding gene sequence (SEQ ID NO: 5), primers were designed, and the antigenic protein Rv0585c was expressed and purified using a prokaryotic expression system (such as Escherichia coli).
Embodiment 2
[0046] Example 2 Synthesis of Mycobacterium tuberculosis antigenic protein Rv0585c T cell epitope peptide
[0047] Based on the T cell IFN-γ release technology, the T cell epitope on the Rv0585c coding gene was predicted by using the bioinformatics software TE predict and IEDB, and the epitope peptide was synthesized by the solid-state synthesis method, and then the tuberculosis patients, Specific T lymphocytes in patients with other lung diseases and healthy people were detected, so as to evaluate the sensitivity and specificity of the antigen for tuberculosis detection.
[0048] The Mycobacterium tuberculosis antigenic protein Rv0585c T cell epitope peptide provided in this example is selected from P24, P26, and P31, and its amino acid sequences are respectively shown in SEQ ID NO: 1-3.
Embodiment 3
[0049] The preparation of embodiment 3 tuberculosis T-SPOT detection kit
[0050] The basic composition of the test kit is as follows:
[0051] ① The protein antigen Rv0585c prepared in Example 1 and / or the epitope peptide antigen synthesized in Example 2: the epitope peptide is selected from at least one of P24, P26, and P31.
[0052] ② Primary antibody: mouse IgG monoclonal antibody against human or animal IFN-γ.
[0053] Enzyme-labeled reagent: another mouse IgG monoclonal antibody labeled with horseradish peroxidase against different epitopes of human or animal IFN-γ.
[0054] ③Standard product:
[0055] Culture plate: 96-well microwell reaction plate containing PVDF membrane or nitrocellulose membrane, positive control wells contain tuberculosis non-specific stimulating antigen (such as PHA, etc.), negative control wells contain PBS or base solution.
[0056] ④ Other reagents and consumables required for T-SPOT detection.
[0057] The primary antibody was immobilized ...
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