[Prokaryotic expression and identification of PPE15 protein from Mycobacterium tuberculosis and preparation of rabbit polyclonal antibodies].

Objective To clone, express and purify PPE15 recombinant protein from Mycobacterium tuberculosis (H37Rv), as well as prepare and characterize its rabbit polyclonal antibody. Methods By The PPE15 gene was amplified from the genome of Mycobacterium tuberculosis H37Rv by PCR, and the His-tagged prokaryotic PPE15 prokaryotic expression plasmid pET28a-PPE15 was constructed by homologous recombination cloning technique, and transformed into E. coli BL21 (DE3). PPE15 expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Recombinant PPE15 was identified by SDS-PAGE, and further purified by affinity chromatography with a Ni-NTA column. The renaturation purified PPE15 protein was used to immunize New-Zealand rabbit to prepare polyclonal antibodies. The antibody specificity was analyzed by Western blot analysis, and antibody titer was determined by indirect ELISA. Results Recombinant prokaryotic PPE15 protein was successfully expressed and purified with a molecular weight of 38 kDa. The purified PPE15 protein exhibited positive reaction with the serum of TB patients and the PPE15 protein, the titer of the polyclonal antibodies reaches more than 1:1 300 480. Conclusion The recombinant protein PPE15 was successfully expressed and purified, and high titer rabbit-derived polyclonal antibody was prepared which provided an experimental basis for further functional studies of PPE15 protein.