5种结核分枝杆菌Rpf样蛋白的活性鉴定及促生长作用

目的探讨结核分枝杆菌5种Rpf样蛋白(RpfA、RpfB、RpfC、RpfD、RpfE)的生物学活性及其促生长作用。方法根据GenBank中结核分枝杆菌H37Rv基因组核酸序列分别设计5种Rpf样蛋白特异性引物序列,采用PCR技术扩增目的片段,克隆、表达及纯化结核分枝杆菌的5种Rpf样蛋白,应用获得的Rpf样蛋白对藤黄微球菌、BCG和结核分枝杆菌标准菌株H37Rv的促生长活性观察。结果成功获得了纯化的五种Rpf重组蛋白,其浓度分别为374.76μg/mL、300μg/mL、116.27μg/mL、306.9μg/mL、349.8μg/mL;促生长效果观察,其中RpfC无明显的促生长活性,RpfE的促生长活性最明显;A、B和D均有一定程度的促进这三种菌生长增殖的作用。结论Rpf样蛋白具有促进结核分枝杆菌增殖的作用,有可能成为一种新型的培养基添加剂,为结核菌的快速培养及相关研究奠定基础。

Prokaryotic expression and biological activities of the recombinant resuscitation promoting factor-like proteins in Mycobacterium tuberculosis

To investigate the biological activities of the resuscitation promoting factor(TPF)-like proteins and its role to promote the growth of microorganisms,5 specific primers for TPF-like protein genes encoding RpfA,RpfB,RpfC,RpfD and RpfE were designed according to the genomic sequences of Mycobacterium tuberculosis H37Rv strain,and these genes were amplified using PCR and cloned into prokaryotic expression vector pGEX-4T-1.Then the recombinant plasmid was transformed to E.coli DH5α and identified by PCR.After induction with IPTG,the recombinant RPFs were purified.The growth-promoting activities to microorganisms(Micrococcus leteus,M.tuberculosis H37Rv and BCG)of the obtained Rpf-like proteins were observed.In this way,5 purified recombinant Rpf proteins were successively obtained with concentrations at 374.76 μg /mL,300 μg /mL,116.27 μg /mL,306.9 μg/mL and 349.8 μg /mL respectively for RpfA.RpfB,RpfC,RpfD and RpfE.As to the growth-promoting effect of these recombinant proteins to 3 microorganisms,all of them except RpfC showed definitely visible growth-promoting effect,in which the most prominent growth-promoting effect occurred in the action of EpfE.It is concluded that Rpf-like proteins process the ability to promote growth of mycobacteria and other microorganisms,therefore,they can be used as a novel additive in culture medium so as to provide basis for the rapid cultivation of mycobacteria and the related researches.