Molecuar cloning of Rv0405c virulence gene in Mycobacterium tuberculosis and its expression in Escherichia coli

Objective To explore the biological functions of the efflux apparatus with cloning and expression of Rv0450c gene of Mycobacterium turberculosis(M.turberculosis) H37Rv strain.Methods The location and in/out orintation of MMPL4 were analyzed by TMHMM and HMMTOP program.Full length Rv0450c gene and 2 fragments,mmp14-161 and mmp14-371,which encoding an inner(161 amino acids) and an outer membrane region(371 amino acides) of MMPL4 were amplified by PCR from genome of M.tuberculosis H37Rv strain and inserted into prokaryotic expression vector pET28b.The recombinant plasmids were confirmed by double digestion with the enzymes and the DNA sequence were transformed into Escherichia coli BL21 (DE3) pLysS strain and induced by isoprogyl B-D-thiogalactopyranoside(IPTG).The expression of the recombinant protein with 6×histidine(His) tag was identified by sodium dodecyl sulfate-polyacrylamide gel electrophosesis(SDS-PAGE) and was purified through the 6 × His affinity chromatography method.Results The size of the constructed recombinant plasmids digested by restricted enzymes of Nhe Ⅰ and Hind Ⅲ was coincident with the expected size.The inserted target gene and its reading frame were coincident w ith the expression vectors.Expressions of the full legnth MMPL4 and the small inner membrane region were low,while the small outer membrane region was overexpressed in E.coli,but as inclusion bodies.Fruther purification of the intact MMPL4 protein resulted in a purification of a much smaller degradative product.Conclusion The expressions of intact of MMPL4 protein and inner/outer membrane region in E.coli were analyzed,which may facilitate fruther functional study of the Rv0450c gene.