结核分枝杆菌叶酸代谢相关基因Rv0992c的性质及功能研究

发布时间：2018-01-05 21:45

本文关键词：结核分枝杆菌叶酸代谢相关基因Rv0992c的性质及功能研究　出处：《西南大学》2011年硕士论文　论文类型：学位论文

【摘要】：结核病严重危害人类的生命健康,其致病菌结核分枝杆菌(Mycobacterium tuberculosis)感染过全世界近三分之一的人口。随着人们对结核分枝杆菌生物学特征及生理代谢的了解,相继研发了链霉素、异烟肼、利福平等治疗性药物,但是这些药物的不合理利用引起耐药分枝杆菌的出现。结核病控制工作中急需新型药物的出现,所以寻找新的结核药物作用靶点及开发新型抗结核药物己成为当前的热点和难点。这就需要加深对结核分枝杆菌生理代谢的了解。叶酸代谢是细菌不可或缺的生理过程,其代谢途径中所涉及的某些酶的编码基因在结核分枝杆菌中还不是很清楚。本研究通过生物信息学方法对M. tuberculosis H37Rv的未知功能基因Rv0992c的蛋白产物进行结构和功能的分析及预测,并对该基因进行克隆、表达及蛋白产物酶学活性研究。从之前对深红红螺菌(Rhodospirillum rubrum)的研究得知Rru_A1084基因编码5-甲酰基-四氢叶酸环连接酶(5-formyltetrahydrofolate cyclo-ligase,也称为次甲基-四氢叶酸合成酶,MTHFS)。M. tuberculosis中Rv0992c基因是Rru A1084的同源物(二者的氨基酸序列具有32.7%的相似性和21.8%的同源性),所以推测前者编码结核分枝杆菌的5-甲酰基-四氢叶酸环连接酶。从GenBank数据库中获得M. tuberculosis H37Rv的Rv0992c的核苷酸序列,设计对引物,以M. tuberculosis H37Rv全基因组为模板,PCR扩增获得Rv0992c基因。通过TA克隆,将PCR产物连接到pMD19-T Simple Vector上,之后亚克隆至载体pET28上,经菌落PCR,质粒酶切以及核苷酸序列测序证明了成功的构建了重组质粒pET28-Rv0992c。重组质粒转化Escherichia coli BL21 (DE3),以温度、时间和IPTG浓度建立三因素三水平的正交实验,优化该重组蛋白质诱导表达条件。以最佳表达条件(0.8mM的IPTG,25℃下诱导4h)进行扩大培养,Ni+凝胶亲和层析柱纯化获得高纯度的重组Rv0992c蛋白。纯化蛋白的酶学活性通过反应产物5,10-次甲基-四氢叶酸在355nm处吸光度的变化得到证实。运用Vector NTI 9, Swiss Model等生物信息学资源对结核分枝杆菌Rv0992c编码蛋白的基本性质进行了分析,包括对蛋白质等电点和分子量的预测,二级结构以及三级结构的预测分析。结果表明：M. tuberculosis H37Rv Rv0992c基因编码蛋白分子量为21412.95Da,理论PI为9.40；二级结构是由3个α螺旋8个β折叠及无规则卷曲构成的。在一二级结构分析的基础上,利用同源建模的方法完成了其三维结构的建模。分析该蛋白在分枝杆菌属中的分布情况,结果显示M. tuberculosis H37Rv的MTHFS同已报道的其他分枝杆菌属来源的MTHFS有较高的同源性。综上,本研究揭示了M. tuberculosis H37Rv Rv0992c序列编码蛋白具有将5-甲酰-四氢叶酸转化为5,10-次甲基-四氢叶酸的生物学功能。
[Abstract]:Tuberculosis serious harm to human health, the pathogenic bacteria of Mycobacterium tuberculosis (Mycobacterium tuberculosis) infection all over the world nearly 1/3 of the population. With the understanding of the biological characteristics of Mycobacterium tuberculosis bacilli and physiological metabolism, have developed streptomycin, isoniazid, rifampin therapeutic drugs, but not the rational use of these drugs cause the emergence of drug-resistant Mycobacterium tuberculosis. The control work in urgent need for new drugs, so looking for new TB drug targets and the development of new anti tuberculosis drugs have become the hot and difficult. It is necessary to deepen the understanding of the physiological metabolism of Mycobacterium tuberculosis. Folate metabolism is a physiological process of bacterial gene encoding some indispensable. The enzyme involved in the metabolic pathway in Mycobacterium tuberculosis is not very clear.
In this study, analyze and predict the structure and function by bioinformatics analysis of Rv0992c protein of unknown function gene M. of tuberculosis H37Rv, and to clone the gene expression and activity of protein enzyme. From the previous of Rhodospirillum rubrum (Rhodospirillum rubrum) research that Rru_A1084 gene encoding 5- formyl tetrahydrofolate ring ligase (5-formyltetrahydrofolate cyclo-ligase, also known as methylene tetrahydrofolate synthetase, MTHFS) Rv0992c.M. tuberculosis Rru gene is the homolog of A1084 (two amino acid sequence homology with 21.8% similarity and 32.7%), so that the former encoding Mycobacterium tuberculosis 5- formyl tetrahydrofolate ligase ring. Nucleotide sequences obtained M. tuberculosis H37Rv Rv0992c from the GenBank database, a pair of primers were designed with M. tuberculosis H37Rv. The genome as a template, PCR Rv0992c gene was amplified by TA. The PCR products were cloned, connected to pMD19-T Simple Vector, then cloned into the vector pET28. By colony PCR, restriction enzyme analysis and nucleotide sequencing proved the successful construction of the recombinant plasmid pET28-Rv0992c. recombinant plasmid was transformed into Escherichia coli BL21 (DE3), with temperature orthogonal experiment, time and concentration of IPTG to establish three levels of three factors, the optimization of recombinant protein expression conditions. With the optimized expression conditions (0.8mM IPTG, 25 DEG C under the induction of 4h) to expand the training, column chromatography to obtain high purity of recombinant protein Rv0992c affinity gel. Ni+ enzymatic activity of purified protein by reaction products 5,10- - methylene tetrahydrofolate in the change of absorbance at 355nm was confirmed.
The use of Vector NTI 9, Swiss Model and other bioinformatics resources for encoding Rv0992c protein of Mycobacterium tuberculosis and the basic properties are analyzed, including the prediction of protein molecular weight and isoelectric point, analysis of two level structure and prediction of three level structure. The results show that the M. tuberculosis H37Rv Rv0992c gene encoding protein molecular weight is 21412.95Da. The theory of PI is 9.40; the two stage structure is folded by 3 alpha helix 8 beta and random coil structure. Based on the analysis of one or two level structure, using homology modeling method to complete the modeling of the three-dimensional structure. Analyzing the distribution of protein in Mycobacterium species, showed homology there are other high M. of Mycobacterium tuberculosis H37Rv MTHFS reported from MTHFS. In conclusion, this study reveals that the M. tuberculosis H37Rv Rv0992c sequence encoding protein with 5- formyl - The transformation of four hydrofolate is the biological function of 5,10- methylene dihydrofolate.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

【共引文献】