Prokaryotic Expression Three Resuscitation-Promoting Factors From Mycobacterium Tuberculosis And Their Preliminary Application

Objective: To construct three prokaryotic expression plasmids carrying Mycobacterium tuberculosis RpfB,RpfD,RpfE gene respectively , express the recombinant proteins in E.coli and observe their biological activity.Methods: : RpfB gene, RpfD gene, RpfE gene from BCG genome were amplified by PCR,and then cloned into pET32a(+) vector. After identified by restriction digestion and DNA sequencing, the constructed recombinant plasmids were transformed into E.coli BL21 respectively. The recombinant proteins were expressed by E.coli BL21 after IPTG induction, identified by Western-blot and purified by affinity chromatograghy.The three recominant proteins(RpfB,RpfD,RpfE) were add into the LB medium respectively, and the M.smegmatis was subcultured on the medium by streaking, spreading and pouring respectively. The time for the colony growth appearing was recorded. Finally the slow growth Mycobacteria(Mtb H37Ra, BCG, M. marinum) were subcultured on the L-J medium which contain the recombinant proteins, and the colony growth time was observed.Result: the size of the RpfB gene, RpfD gene, RpfE gene from recombinant plasmids digested by restricted enzymes were 1089bp, 465bp, 519bp respectively. The DNA sequences were the same as the sequences reported on the literatures.A 61KDa, 39KDa and 41KDa proteins were found by SDS-PAGE and Western-blot. M.smegmatis in the LB medium which added anyone of these recominant proteins could grow faster than it in the control medium. As for the slow growth mycobacteria, the test groups has little effect on bacterial growth against the control groups.Conclusion: The RpfB gene,RpfD gene, RpfE gene were successfully amplified and cloned into pET32a(+) vector, and the recombinant proteins were expressed and purified. All the three recombinant proteins had the biological activity. But in the study ,we didn't find that the Rpf-like proteins had the significate activity to enhance the growth of the slow growth mycobacteria. But we should look forward to further study under other conditions.