结核分枝杆菌ClpX和ClpP2蛋白酶的克隆表达及其性质研究

发布时间：2018-01-14 08:29

  本文关键词：结核分枝杆菌ClpX和ClpP2蛋白酶的克隆表达及其性质研究　出处：《西南大学》2010年硕士论文　论文类型：学位论文

  更多相关文章： ClpX蛋白酶 ClpP2蛋白酶 结核分枝杆菌 氧化压力耐受

【摘要】： 结核分枝杆菌所导致的结核病仍然是全球人类健康的主要威胁,结核分枝杆菌每年引起两三百万的人死亡。结核分枝杆菌是一种严格而成功的胞内致病菌,在宿主的免疫系统细胞,主要是巨噬细胞内存活并复制。蛋白酶一直被认为是致病微生物的重要毒力因子。比较基因组学研究发现结核分枝杆菌主要有16大类蛋白酶,其中许多成员都与结核分枝杆菌的致病性和在单核细胞、巨噬细胞中的持留性相关。蛋白质的降解是所有生物生理调控和蛋白质量控制所必需的。弄清蛋白质降解系统对细胞内蛋白质质量的控制,揭示致病菌在宿主免疫压力下细胞内蛋白质稳态的维持机制,有利于进一步揭示结核分枝杆菌的致病机理,进而为药物开发提供可能的靶标。 结核分枝杆菌入侵人体和在巨噬细胞中持留性感染时往往会遭遇许多不利的环境压力,诸如营养缺乏、低PH值、氧化压力及高温压力等。其中,氧化压力和热应激条件下会产生大量蛋白质的解折叠和聚集,如何降解这些错误折叠的蛋白质,对维持结核分枝杆菌在压力条件下的正常生长起着至关重要的作用。细菌胞内蛋白的水解由四种依赖ATP的蛋白酶执行,分别是ClpXP,ClpYQ(HslUV),FtsH和Lon家族。在结核分枝杆菌中未发现HslUV和Lon蛋白家族,因此ClpXP蛋白酶在结核分枝杆菌中的功能显得更加重要。 为了研究结核分枝杆菌可能的ClpX和ClpP2蛋白的基本功能,本研究提取结核分枝杆菌H37Rv的基因组,采用体外PCR扩增的方法得到clpX(Rv2457c)和clpP2(Rv2460c)基因。将clpX和clpP2基因的PCR产物分别连入载体pET28a(+)和pET32a(+)中,经菌落PCR及测序证明成功构建了重组质粒pET28-clpX和pET32-clpP2。将重组质粒转化大肠杆菌BL21(DE3),IPTG诱导表达重组蛋白,Ni亲和层析纯化重组蛋白,并进行了ClpX蛋白酶的ATP酶活的体外初步检测。为了进一步了解ClpXP蛋白在生物体内的活性,我们研究了过表达ClpXP蛋白酶对宿主菌的影响,包括：①过表达ClpX和ClpP2蛋白酶对宿主菌生长的影响；②过表达ClpX和ClpP2蛋白酶体对宿主氧化应激的影响。研究证明ClpX蛋白的过表达促进了宿主菌的生长,共表达ClpX和ClpP2蛋白也能促进宿主菌的生长,ClpP2蛋白的过表达能够增加宿主细胞对过氧化氢的耐受。长时间过表达ClpP2蛋白酶,纯化到的ClpP2蛋白酶具有分子量大小有差异的两条目的蛋白条带,MALDI-TOF-TOF鉴定结果显示两条大小有差异的目的蛋白均为ClpP2重组蛋白。生物信息学分析显示ClpP2蛋白酶存在酰胺化修饰位点、CAMP磷酸化位点等多个修饰位点,可能是由于这些位点的修饰导致了蛋白质分子量的变化。并用在线数据库预测了ClpX和ClpP2蛋白可能的作用网络,进一步分析了ClpXP蛋白酶在细胞内的生理功能。 从实验结果来看,结核分枝杆菌可能的ClpX和ClpP2蛋白在细菌生长和氧化压力耐受中发挥着很重要的作用。对它们的深入研究可以揭示结核分枝杆菌对宿主氧化压力耐受和体内蛋白质质量控制的机制,为我们开发新的抗结核靶标提供思路。
[Abstract]:Caused by Mycobacterium tuberculosis is still a major threat to human health worldwide, Mycobacterium tuberculosis caused by two or three million people each year. The death of Mycobacterium tuberculosis is a kind of strict and successful intracellular pathogen in the cells of the host immune system, mainly macrophages survive and replicate. Protease has been considered the important virulence factors of pathogenic microorganisms. Comparative genomics of Mycobacterium tuberculosis found there are 16 main types of protease, many of the members are related to the pathogenicity of Mycobacterium tuberculosis and on monocytes and macrophages in the retention. The degradation of protein is required for all physiological regulation and protein quality control. The quality of protein in cells to protein degradation, reveal the pathogen in the host immune pressure cell protein homeostasis mechanisms conducive to One step reveals the pathogenesis of Mycobacterium tuberculosis and provides a possible target for drug development.
Mycobacterium tuberculosis infection to invade the body and stay in macrophages will often encounter many adverse environmental stresses, such as lack of nutrition, low pH, oxidative stress and high temperature pressure. Which will produce a large number of protein unfolding and aggregation of oxidative stress and heat stress conditions, the degradation of these misfolded proteins. To maintain normal growth of Mycobacterium tuberculosis under pressure plays a vital role. The hydrolysis of intracellular bacterial protein by four ATP dependent proteases, including ClpXP, ClpYQ, FtsH and Lon (HslUV) family. HslUV and Lon proteins were found in Mycobacterium tuberculosis, so the function of ClpXP protease in Mycobacterium tuberculosis is becoming more and more important.
In order to study the basic function of Mycobacterium tuberculosis may ClpX and ClpP2 protein, the extraction of Mycobacterium tuberculosis H37Rv genome, using the method of in vitro amplification of PCR clpX (Rv2457c) and clpP2 (Rv2460c) gene. The product of PCR clpX and clpP2 gene were inserted into vector pET28a (+) and pET32a (+) in the colony PCR and sequencing proved that recombinant plasmid pET28-clpX was successfully constructed and the recombinant plasmid pET32-clpP2. was transformed into E. coli BL21 (DE3), recombinant protein expression was induced by IPTG, purified recombinant protein Ni affinity chromatography, and has carried on the preliminary detection of ATP enzyme ClpX protease activity in vitro. In order to further understand the activity of the ClpXP protein in organism inside, we investigated the effects of overexpression of ClpXP protein on host bacteria, including: the impact of ClpX overexpression and ClpP2 protease on the growth of the host bacteria; the expression of ClpX and ClpP2 proteasome in night The main effect of oxidative stress. The study shows that overexpression of ClpX promotes the growth of the host bacteria, co expression of ClpX and ClpP2 protein can also promote the growth of the host bacteria, overexpression of ClpP2 can increase the tolerance of host cells to hydrogen peroxide. Long time overexpression of ClpP2 protease, the purified ClpP2 protease with molecular weight there are differences between the two protein bands, the identification results of MALDI-TOF-TOF showed that two size different purpose protein was the recombinant ClpP2 protein. Bioinformatics analysis showed that ClpP2 protease exists amidation sites, multiple modification sites CAMP phosphorylation sites, may be due to modification of these sites leads to changes in protein molecular weight. And forecast the network ClpX and the role of ClpP2 protein in online databases, further analysis of the physiological function of ClpXP protease in cells.
From the experimental results, Mycobacterium tuberculosis ClpX and ClpP2 protein may play an important role in bacteria growth and oxidative stress tolerance. On their further study can reveal the Mycobacterium tuberculosis on host oxidative stress tolerance and protein quality control system, we provide ideas for the development of new anti tuberculosis targets.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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