Cloning, Expression And Its Function Of The Rv1494-Rv1495 Gene Of Mycobacterium Tuberculosis

Tuberculosis is still the world's leading cause of mortality owing to an infectious bacterial agent, Mycobacterium tuberculosis(MTB). It is estimated that one third of the world's population were infected with M. tuberculosis,however,most of new cases were caused by the latent M. tuberculosis been actived.Althought the chem schedule does great work in treating active tuberculosis,it does little in treating latent. More and more scientists focus on the research of M. tuberculosis. So it is urgent and important for us to study the correlate genes and the mechanisms of the M tuberculosis.There are many documents indicated many kinds of pathogenic bacteriums have the ability to lead latent infection, maybe which is related with the genome conservatived TA system of prokaryote.The TA system is made up of toxin and antitoxin and is concerned to be quality control system of prokaryote.This system include seven families of two members (ccd,relBE,parDE,higBA,mazEF,phd/doc,vapBC) and one family of three members(ω—ε—ξ),there are many similars among the proteins of those families. Gerdes found 38 TA loci in H37Rv strain of M tuberculosis by bioinformation and there were 9 mazEF loci. Central to understanding the TA families are related with the latent infection of pathogenic tuberculosis,we choose one family (Rv1494-Rv1495) from the 9 mazEF loci.With this in mind, we analyzed two proteins from the Rv1494 and Rv1495 by bioinformation and found protein of Rv1494 and Rv1495 shared similarly motifs with the family of MazEF in E.coli. We amplified the gene Rvl494 and Rvl495 from the genome of H37Rv for the first time and the genes were subcloned into expression vector pET32a(+) to express its proteins.This provided the basis for the further study of the gene Rv1494-Rv1495. At the same time,the Rv1494-Rv1495 fuse gene was amplified and subcloned into E.coli-Mycobacterium shuttle vector pMV261. The recombinant vector was transformed into M. smegmatis by electroporation. The transforms were induced to express a predicted protein under heat pressure. We compare the M. smegmatis which contained recombinant vector with the M. smegmatis contained vector pMV261 in growing curve,heat pressure and acid pressure.In summary, the recombinant expression plasmids pET-Rv1494 and pET-Rv1495 were successfully constructed firstly and the fusion proteins were obtained from the induced E. coli BL21.The recombinant vector pMV261-(Rv1494-Rv1495) was construct and transformed into M. smegmatis by electroporation. Its fuction of the transforms were researched initially. The result suggests a possible role for Rv1494-Rv1495 in the resistancer outer pressure for bacterial latent infection.