结核分枝杆菌Rv1494-Rv1495基因的克隆表达及功能研究

发布时间：2018-05-27 05:40

本文选题：结核分枝杆菌 + Rv1494　；参考：《四川大学》2007年硕士论文

【摘要】： 结核病是由结核分枝杆菌(mycobacterium tuberculosis，MTB)引起的一种古老的传染病，目前仍是世界上引起死亡的最主要的疾病之一。目前，全球大约有1／3的人口有潜伏期结核分枝杆菌的感染，而大部分新的结核病例是由潜伏期的结核分枝杆菌的活化引起的。虽然结核病的化疗方案在结核病的控制中起了很大作用，但其主要对活动性结核病起作用，对潜伏期的结核分枝杆菌的感染则作用不大。因此，了解MTB持留的机制，研究其持留相关基因，，将对结核病的治疗意义重大。研究结果提示，很多不同种属的病原菌都具有形成潜在感染的能力，可能与原核生物染色体基因组上保守的TA系统有关，其主要由毒素和抗毒素位点组成，被认为是原核生物基因表达的质量控制系统。该系统包括7个两组分基因家族(ccd、re1BE、parDE、higBA、mazEF、phd／doc、vapBC)和1个三组分家族(ω—ε—ξ)，这些家族间蛋白序列表现出较高的相似性，并且有各自的模块。Gerdes等通过生物信息学方法发现在结核杆菌H37Rv株中含有38个TA loci，其中包括9个mazEF位点。为研究TA家族与结核杆菌潜伏感染之间的关系，我们选取了其中的一组可能的mazEF位点Rv1494和Rv1495。经过生物信息学分析，Rv1494和Rv1495基因表达的蛋白所具有的模块与TA家族中的常见模块一致。我们首先从基因组中扩增出这两个基因片段，并在原核融合表达载体pET32a(+)中成功表达了相应的融合蛋白。同时我们还克隆了Rv1494-Rv1495融合基因，并将其重组到穿梭表达载体pMV261中，经电转化导入无毒的耻垢分枝杆菌(mc~2155)中，并在重组耻垢分枝杆菌(Rmc~2155)中成功表达了11KD的Rv1494-Rv1495编码蛋白。本研究还对负载空质粒pMV261的耻垢分枝杆菌及负载重组质粒的耻垢分枝杆菌在生长曲线、酸压力及热压力方面作了比较。综上所述，本研究首次对结核分枝杆菌Rv1494和Rv1495基因进行了克隆和表达，同时构建了重组的穿梭质粒pMV261-(Rv1494-Rv1495)，并将其转化入耻垢分枝杆菌中，对重组耻垢分枝杆菌MCEF功能进行了初步的研究。结果提示，Rv1494-Rv1495可能对结核杆菌在抵抗外界压力以维持其潜伏感染中有一定作用。
[Abstract]:Tuberculosis (TB) is an ancient infectious disease caused by Mycobacterium tuberculosisus MTB (Mycobacterium tuberculosis), and it is still one of the leading causes of death in the world. At present, about a third of the world's population has a latent mycobacterium tuberculosis infection, and most of the new cases of tuberculosis are caused by the activation of the latent mycobacterium tuberculosis. Although the chemotherapy regimen of tuberculosis plays a great role in the control of tuberculosis, it plays an important role in active tuberculosis, but it has little effect on the infection of Mycobacterium tuberculosis in latent period. Therefore, understanding the mechanism of MTB retention and studying its retention related genes will be of great significance in the treatment of tuberculosis. The results suggest that many pathogens of different species have the ability to form potential infections, which may be related to the conserved TA system on the chromosomal genome of prokaryotes, which is mainly composed of toxin and antitoxin sites. It is considered to be a quality control system for gene expression in prokaryotes. The system consists of seven two-component gene families, the two component gene family, the BCCfamily of two components, the protein sequence of these families shows high similarity, and one family of three components (蠅-蔚-尉), which shows high similarity in the protein sequence between the two families, and the protein sequence of the two families is similar to that of the protein family. By bioinformatics method, 38 TA locions were found in Mycobacterium tuberculosis H37Rv strain, including 9 mazEF loci. To study the relationship between TA family and latent infection of Mycobacterium tuberculosis, we selected one of the possible mazEF loci Rv1494 and Rv1495. Bioinformatics analysis showed that the proteins expressed in Rv1494 and Rv1495 genes had the same modules as those in the TA family. We first amplified the two fragments from the genome and successfully expressed the fusion protein in the prokaryotic fusion expression vector pET32a (). At the same time, we cloned the Rv1494-Rv1495 fusion gene, and recombined it into the shuttle expression vector pMV261, and then transformed it into the nontoxic Mycobacterium smearicum mcc2155), and successfully expressed the Rv1494-Rv1495 coding protein of 11KD in the recombinant Mycobacterium smearicum Rmcn2155). The growth curve, acid pressure and thermal pressure of Mycobacterium pubescens loaded with empty plasmid pMV261 and recombined plasmid were compared. In conclusion, the Rv1494 and Rv1495 genes of Mycobacterium tuberculosis were cloned and expressed for the first time, and the recombinant shuttle plasmid pMV261-nrv1494-Rv1495 was constructed and transformed into Mycobacterium smeareus. The MCEF function of recombinant Mycobacterium smegmatis was preliminarily studied. The results suggest that Rv1494-Rv1495 may play a role in resisting external pressure and maintaining latent infection of Mycobacterium tuberculosis.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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