CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR WITH Rv0901 GENE,A IMAGINABLE VIRULENCE GENE OF MYCOBACTERIUM TUBERCULOSIS

Objective To construct the eukaryotic expression vector with Rv0901 of Mycobacterium tuberculosis and observe the expression of Rv0901 protein for exploring the function of Rv0901 gene. Methods Rv0901 gene was amplified by PCR from genome of M. tuberculosis and inserted into eukaryotic expression plasmid pcDNA3.1(+) followed by digestion with KpnⅠ and EcoRⅠ. The recombinant plasmid pcDNA3.1-Rv0901 was transformed into E. coli DH5α and selected with ampicillin. The positive clones containing the recombinant plasmid pcDNA3.1-Rv0901 were verified by restriction endonucleases KpnⅠ and EcoRⅠ and PCR. Sequence analysis was performed to verify the recombinant plasmid. The recombinant plasmid pcDNA3.1-Rv0901 was transfected into Cos7 cells, RT-PCR was performed to detect the effect of transfection. Extract the total protein of transfected cells and collect the cultural supernatant, the expression of Rv0901 protein was identified by SDS-PAGE and Western-blotting. Results and Conclusion The eukaryotic vector for Rv0901 gene has been successfully constructed. The 19 ku Rv0901 protein was successfully expressed in Cos7. It provided the basis for the further study of gene Rv0901.