Cloning, Prokaryotic Expression And Biological Functions Of Mycobacterium Tuberculosis Rv1884c And Rv0867c Genes

Tuberculosis(TB) is a chronic respiratory infectious disease caused by pathogen Mycobacterium tuberculosis(MTB). It severely threats the public health and kills more people in the world than any other single bacterial infection. In the past 10 years,accompanying the increasing incidence of AIDS, the incidence and mortality of TB are increasing rapidly in many countries and districts and becoming the main cause of death. It is estimated that one-third of the world population are infected with MTB and most of them harbor a persistent (latent) infection. There appear about 8 millions people infected with TB and the number of death is about 2 millions annually. From 2002 to 2020, if the infection with MTB could not be controlled effectively, there would be another 1 billion people infected with MTB. China is one of the 22 countries with high incidence of TB, ranks the second place in the world. This is due to the TB multidrug-resistant strains, co-infection with HIV and the increasing mobility of population. About 90% TB patients are dormantly infected by MTB. This urgent situation requires through and in-depth research on TB pathogenesis in order to find more effective new techniques, new methods and new vaccines for detection, treatment, as well as protection against mycobacterial infection. Micrococcus leteus secretes a small protein called Resuscitation- promoting factor (Rpf), which has autocrine and paracrine signaling function and is required for the resuscitation of dormant cells. Rpf is essential for growth of Micrococcus leteus. In picomolar concentrations, it increases the viable cell count of dormant Micrococcus leteus and several other high G+C Gram- positive organisms including Mycobacterium avium, Mycobacterium bovis(BCG), Mycobacterium kansasii, Mycobacterium smegmatis, and Mycobacterium tuberculosis. Moreover, scientists have revealed genes homologous to Micrococcus leteus Rpf in a vaiety of other GC-rich bacterias, including Streptomyces coelicolor, Corynebacterium glutamicum, Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis by bioinformatical methods. All of these proteins are termed as bacterial cytokines because they have the same function resembling the eukaryotic cytokine. Mycobacterium tuberculosis has five genes with regions of high homology to the Micrococcus leteus Rpf. The Rpf-like genes of Mycobacterium tuberculosis H₃₇Rv are distributed throughout the chromosome,with gene designations Rv0867c(RpfA),Rv1009(RpfB),Rv1884c(RpfC), Rv2389c(RpfD) and Rv2450c(RpfE). Rpf-like protein may play an important role in the reactivation of quiescent bacilli. Individual Rpf-like genes can be deleted from the chromosome of MTB with no significant alteration of growth in liquid culture. It indicates that the function of the various MTB Rpfs may be entirely or partially redundant. But the in vitro expression kinetics of the five MTB Rpf-like genes falls into different pattern. In our work Rv1884c and Rv0867c genes were cloned and expressed in E.coli DH5α.The biological function of the two purified proteins were investigated.1. Cloning,prokaryotic expression and the purification of Mycobacterium tuberculosis Rv1884c and Rv0867c genesBased on MTB Rv1884c and Rv0867c nucleotide sequences in GenBank, specific primers were designed using the software of Primer Premier 5.0. Then the genome DNA of MTB was extracted and the two fragments were amplified by PCR. The two sequences were cloned into EcoRâ… ,BamHâ… and HindIII sites of pGEX-4T-1 and pUC19 cloning vectors. After simple identification, the two fragments of right size were subcloned into expression vector pGEX-4T-1 and pPRO-EXHT. After confirmed by DNA sequencing, the correct recombinant plasmids were transformed into E.coli DH5α. The two recombinant bacterias were induced at 35℃and 37℃seperately using 0.1M IPTG. The recombinant fusion proteins pGEX-4T-1-Rv1884c and pPro-EXHT-Rv0867c were analysed by SDS-PAGE and their molecular weight are 45KD and 80KD respectively. The two recombinant proteins were purified with GSTrap FF affinity chromatography column and Ni2+-NTA column.2. Preliminary study on the biological function of proteins Rv1884c and Rv0867cCertain concentration of Rv1884c and Rv0867c proteins were added into the Micrococcus leteus, BCG and MTB culture medium. The results showed that Rv1884c and Rv0867c proteins could both stimulate the growth of Micrococcus leteus, BCG and MTB. Moreover, Rv0867c is more potent than Rv1884c at the same concentration.In summary, the two Rpf-like genes were cloned , expressed and purified successfully. They both have biological activity and can promote the growth of Micrococcus leteus, BCG and MTB.