| Tuberculosis (TB)is a chronic respiratory infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB). In recent years,such factors as no enough efficiency of BCG,the increasing frequency of drug-resistance MTB,poverty,incidence of HIV-associated infection and increase of population migration,have made TB become a worldwide disease. BCG, which is currently the only vaccine against TB, shows little efficacy on pulmonary TB of adults. So it is significant to research on its pathogenic and immune mechanism for new diagnosis methods and vaccines to prevent TB.Resuscitation-promoting factor (Rpf) of Micrococcus luteus is the first discovered bacteria's growth factor that can promote the resuscitation of dormant cells and stimulate the growth of bacteria. Genome sequencing has discovered that there exist more than 30 homologous proteins in other Gram-positive bacteria, such as Mycobacterium leprae, Mb.tuberculosis, M.bovis, Streptomyces spp and Corynebacterium glutamicum. It is revealed that the genome of MTB can encode 5 Rpf-like proteins, including Rv0867C(Rpf A),Rv1009(Rpf B),Rv1884C(Rpf C),Rv2389C(Rpf D)and Rv2450C(Rpf E), which are speculated to have the funcation of promoting the growth of bacteria. Furthermore, all of the proteins contain conserved segments of 70 amino acid residues, which are important for the resuscitation of Rpf and have the same biological function with Rpf proteins. It is further found that the domains contain a highly conserved glutamate residue(Glu54), which is of importance for the resuscitation activity and coincides with the catalytic activity of lysozyme.AIM:To efficiently express the Rpf domain of Micrococcus luteus and its mutant proteins in prokaryotic cells and investigate the biological activities.METHODS AND RESULTES:1. Expression and purification of Rpf domain and its mutant proteinsThe genes of Rpf domain and its mutants (E54K, E54A) were amplified by PCR from the genome of Micrococcus luteus and cloned into the pMD18-T vector. It was demonstrated that the sequences of PCR product were identical with those in GenBank and the mutable sites accord with enactment. After sequenced, the Rpf domain and its mutant genes were subcloned into expression vector PGEX-4T-1. The recombinant plasmids were transfected into E.coli DH5αstrain and induced with IPTG. The aim proteins were identified by SDS-PAGE analysis and by Western-blot with monoclonal antibodies against Rpf domain (mAb). The SDS-PAGE analysis showed that the fusion proteins of the Rpf domain and its mutants were successfully expressed. The relative molecular mass was consistent with what had been reported,which was also confirmed by Western-blot analysis that there were specific bindings at 32kDa with Rpf domain mAb. Purified the fusion proteins by GS-4B purification system. 2. Biological activity of Rpf domain and its mutant proteins Mycobacterium smegmatis strains were diluted by LB at 1:150. Rpf domain, its mutant proteins and its antibodies were added to M.S with different concentration. the tubes were incubated at 37℃.Taken 200ul from each tubes every four hours to detected the A600nm. The results showed that the purified GST-Rpf domain could stimulate resuscitation of M.S and reduce its growing period. The protein of Rpf domain have the same biological activity with the Rpf protein. E54K has an obvious restraining effect on the growth of M.S, while E54A didn't have the function.CONCLUSIONS:Rpf domain and two kinds of its mutant proteins were obtained. Both the Rpf domain protein and Rpf protein could stimulate the resuscitation of M.S. The promoting effects were counteracted if the antibodies of Rpf and its domain were added. E54K could inhibit the growth of M.S, while E54A didn't have the funcation. The results provide references for investigation of the influence of the Rpf domain and its mutants on the growth of M. luteus and MTB, as well as they may be an appropriate candidate for new diagnostic reagents and subunit vaccine to TB control and prophylaxis. |
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