Eukaryotic expression and identification of resuscitation promoting factor E of Mycobacterium tuberculosis

Objective To construct the eukaryotic expression plasmid of resuscitation promoting factor E of Mycobacterium tuberculosis. Methods Resuscitation promoting factor E gene fragment of Mycobacterium tuberculosis was amplified by PCR. The fragment was cloned into PcDNA 3.1 (-)vector. After sequencing the recombinant plasmid, it was transfected into CHO cells to express resuscitation promoting factor E protein. The expression of cloned gene mRNA in eukaryotic cells was detected by RT-PCR. The protein was collected and purified, and the targeted protein expression was detected by SDS-PAGE analysis and Western blot analysis. The immunogenicity of protein was identified by lymphocyte proliferation assay (CCK-8). Results The eukaryotic expression plasmid of resuscitation promoting factor E of Mycobacterium tuberculosis was successfully constructed. It was confirmed that the cloned gene mRNA was expressed in the eukaryotic cells,the targeted protein was successfully expressed in the eukaryotic cells, and the expressed protein had immunogenicity. Conclusions The eukaryotic expression system of resuscitation promoting factor E was successfully constructed. Key words: Eukaryotic expression; Resuscitation promoting factor E; China hamster ovary cells