Epitope peptide of mycobacterium trberculosis protein antigen, and its application
A technology of expression cassettes and recombinant bacteria, applied in the direction of bacterial antigen components, applications, peptides, etc., can solve problems that have not been reported in research
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Embodiment 1
[0029] Example 1. Acquisition of antigenic epitopes of Mycobacterium tuberculosis recognized by γδT cells
[0030] 1. Construction of tuberculosis-specific and non-specific γδTCR transfected cell lines
[0031] Take 5 ml of heparin anticoagulated fresh venous blood from a tuberculosis patient (obtained from Beijing Chest Hospital, informed by the patient). Add to the test tube that has been added with an equal volume of lymphocyte separation medium in advance to avoid destroying the interface, and centrifuge at 500 × g for 20 minutes; take the white interface layer in the test tube and put it in another test tube, add an equal volume of RPMI-1640 culture medium (Hyclone company, Catalog: SH30809) washed twice. After centrifugation, add Trizol, let stand for 5 minutes, add 200 μl chloroform, shake vigorously, place at room temperature (25°C) for 3 minutes, and centrifuge at 12,000×g, 4°C for 15 minutes. Carefully absorb the upper aqueous phase, transfer to a new centrifuge tu...
Embodiment 2
[0069] Example 2, Functional Identification of Epitope Peptide TP1
[0070] 1. Detection of IL-2 secretion by candidate epitope peptides stimulating tuberculosis-specific γδTCR transfected cells
[0071] Add 500 μl of RPMI-1640 culture solution (Hyclone Company, Catalog: SH30809) containing 10 μg, 20 μg and 40 μg of candidate epitope peptide TP1 (the epitope peptide TP1 obtained in Example 1) to the wells of a 24-well plastic culture plate. Coated and incubated at 37°C for 2 hours. The coating solution was discarded, and washed three times with RPMI-1640 culture medium (Hyclone Company, Catalog: SH30809). 1×10 6 tester cells / ml, 1×10 6 Each / ml driver cells were added to the washed 24-well culture plate. At 24 hours, collect tester cell supernatant containing 10 μg TP1, tester cell supernatant containing 20 μg TP1, tester cell supernatant containing 40 μg TP1, driver cell supernatant containing 10 μg TP1, driver cell supernatant containing 20 μg TP1 supernatant, driver cel...
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