结核分枝杆菌三种复活促进因子的原核表达和初步应用

发布时间：2018-03-01 03:20

本文关键词： 结核分枝杆菌复活促进因子克隆表达纯化　出处：《重庆医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:构建Mtb复活促进因子B(RpfB)、D(RpfD)、E(RpfE)的原核表达质粒,并在E.coli中表达和纯化重组的Rpf样蛋白,初步探讨其对分枝杆菌生长的影响。方法:以BCG基因组为模板,用PCR法扩增出RpfB(1089bp)、RpfD(465bp)和RpfE片段(519bp),并分别连接在pET32a(+)载体上,重组质粒经酶切和DNA测序鉴定后,转化大肠杆菌BL21,IPTG诱导表达了RpfB、RpfD和RpfE蛋白;用免疫印迹法(Western-blot)鉴定重组蛋白后,采用Ni-NTA树脂亲和纯化目的蛋白。然后将三种重组蛋白分别添加到LB培养基中,利用三种不同的方法(划线法、涂布法、倾注法)接种MS,观察不同的重组蛋白对细菌生长的影响。最后,以BCG、Mtb H37Ra、海分枝杆菌为研究对象,将三种重组蛋白分别添加到L-J培养基中,通过观察菌落形成时间了解不同蛋白对细菌生长的影响结果:重组质粒经双酶切鉴定,片段大小与预计相符,测序结果与文献报道一致。重组蛋白经SDS-PAGE分析和Western-blot鉴定均发现分别在61kDa、39kDa、41kDa处有特异性条带。用划线法、涂布法、或倾注法均可观察到LB培养基中到添加了任意一种重组蛋白都能加快MS的生长。将重组蛋白加入到L-J培养基中时,添加蛋白组与对照组比较无显著差异。结论:成功构建了重组表达质粒pET32a(+)-RpfB、pET32a(+)-RpfD、pET32a(+)-RpfE,并获得了3种具有生物学活性的重组蛋白,能促进MS的生长。未能观察到添加了Rpf样蛋白的L-J培养基有明显的促迟缓生长型分枝杆菌(BCG、Mtb H37Ra、海分枝杆菌)生长的作用。
[Abstract]:Objective: to construct the prokaryotic expression plasmid of Mtb resurrection promoting factor (RpfB), and to express and purify the recombinant Rpf like protein in E.coli, and to explore its effect on the growth of Mycobacterium. Methods: using the BCG genome as a template, RpfBn1089bpPNV) and RpfE fragments were amplified by PCR and ligated into pET32a () vector respectively. The recombinant plasmid was digested and identified by DNA sequencing. The recombinant plasmid was transformed into E. coli BL21 IPTG to induce the expression of RpfBU RpfD and RpfE protein. The recombinant protein was identified by Western blot and purified by Ni-NTA resin affinity. Then three recombinant proteins were added to LB medium respectively. The effects of different recombinant proteins on the growth of bacteria were observed. Finally, three recombinant proteins were added to L-J medium with BCGMtb H37Raand Mycobacterium seagrass as the research object. Effects of different proteins on bacterial growth by observing the time of colony formation. Results: the recombinant plasmid was identified by double enzyme digestion, the size of the recombinant plasmid was consistent with the expected size, and the sequencing results were consistent with those reported in the literature. The specific bands of the recombinant plasmid were found to be at 41 kDa by SDS-PAGE analysis and Western-blot analysis, respectively. It was observed that the growth of MS could be accelerated by adding any kind of recombinant protein from LB medium to L-J medium, but there was no significant difference between the added protein group and the control group when the recombinant protein was added to L-J medium. Conclusion: the recombinant expression plasmid pET32a (pET32a) was successfully constructed, and three kinds of recombinant proteins with biological activity were obtained. It was not observed that L-J medium supplemented with Rpf like protein could obviously promote the growth of Mycobacterium lactis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378.91

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