异柠檬酸裂解酶基因克隆与表达特征研究

发布时间：2018-01-03 02:41

本文关键词：异柠檬酸裂解酶基因克隆与表达特征研究　出处：《长春工业大学》2010年硕士论文　论文类型：学位论文

【摘要】：近年来世界范围内结核病发病率明显上升,结核病感染的持留状态这一问题普遍存在。异柠檬酸裂解酶,一种由ICL基因编码,作为乙醛酸代谢途径中的关键酶,在结核分枝杆菌感染的巨噬细胞及其在人体内持续感染起关键支持作用。结核分枝杆菌代谢途径的这一关键酶已经被作为抗结核药物研究和开发的新型靶点,这就需要表达出具有生物学活性的重组异柠檬酸裂解酶。为表达出具有生物活性的重组异柠檬酸裂解酶,本研究以结核分枝杆菌H37Rv基因组为模板,通过PCR反应扩增该菌株的ICL基因,并将测序正确的ICL基因,克隆入原核表达载体pET-28a(+)中,取名为pET28-ic1。重组异柠檬酸裂解酶在大肠杆菌BL21(DE3)中表达出来。通过Ni-NTA亲和层析柱纯化,以获得纯化的结核分枝杆菌H37Rv异柠檬酸裂解酶重组蛋白。对重组蛋白ICL进行酶学性质分析,证明该重组蛋白其具有异柠檬酸裂解酶活性。现已成功在结核分枝杆菌H37Rv中将异柠檬酸裂解酶表达出来,并鉴定得出纯化的重组异柠檬酸裂解酶具有生物活性。为了测定异柠檬酸裂解酶的活性,需要实时测定NADH量的减少。异柠檬酸在异柠檬酸裂解酶催化下形成乙醛酸,乙醛酸可通过乳酸脱氢酶和NADH还原成甘醇酸酯。NADH的减少则通过加入甲佨染料MTS(MTS／PMS比率为100：1),并在490nm处测定吸光度值来确定。纯化的异柠檬酸裂解酶重组蛋白酶活为1.84×102μmol×mg-1×min-1。异柠檬酸裂解酶重组蛋白生物活性的最适PH值为7.4。我们应用原核表达系统,在结核分枝杆菌H37Rv中将ICL克隆和表达出来,纯化出了具有生物学活性的结核分枝杆菌H37Rv可溶性异柠檬酸裂解酶,并对其酶学性质进行了研究,酶学性质鉴定获得了具有生物学活性的重组蛋白。本研究为该酶免疫学研究及新型抗结核药物的开发与设计提供设计方案与理论基础。
[Abstract]:In recent years, the incidence of tuberculosis has increased worldwide, and the persistence of TB infection is widespread. Isocitrate lyase, a gene encoded by ICL. As a key enzyme in glyoxylic acid metabolism pathway. Macrophages infected by Mycobacterium tuberculosis and their persistent infection in humans play a key supporting role. This key enzyme of Mycobacterium tuberculosis metabolic pathway has been used as a new target for the research and development of anti-tuberculosis drugs. This requires the expression of recombinant isocitrate lyase with biological activity. In order to express recombinant isocitrate lyase with biological activity, the ICL gene of Mycobacterium tuberculosis H37Rv was amplified by PCR reaction. The correct ICL gene was cloned into the prokaryotic expression vector pET-28a (). The recombinant isocitrate lyase was expressed in E. coli BL21DE3 and purified by Ni-NTA affinity chromatography. The purified recombinant protein of Mycobacterium tuberculosis H37Rv isocitrate lyase was obtained. The enzymatic properties of the recombinant protein ICL showed that the recombinant protein had isocitrate lyase activity. The isocitrate lyase has been successfully expressed in Mycobacterium tuberculosis H37Rv, and the purified recombinant isocitrate lyase has been identified to have biological activity in order to determine the activity of isocitrate lyase. The amount of NADH needed to be determined in real time. Isocitric acid was catalyzed by isocitrate lyase to form glyoxylic acid. Glyoxylic acid can be reduced by lactate dehydrogenase and NADH to glycolate. Nadh is reduced by adding MTS(MTS/PMS ratio of 100: 1). The purified recombinant protease activity of isocitrate lyase was 1.84 脳 102 渭 mol 脳 mg-1 脳 min-1. The recombinant protein of isocitrate lyase was determined by measuring the absorbance at 490 nm. The optimum PH value of biological activity is 7.4. ICL was cloned and expressed in Mycobacterium tuberculosis H37Rv using prokaryotic expression system. The soluble isocitrate lyase of Mycobacterium tuberculosis H37Rv with biological activity was purified and its enzymatic properties were studied. The recombinant protein with biological activity was obtained by the identification of enzymatic properties. This study provides a design scheme and theoretical basis for the immunological study of the enzyme and the development and design of novel antituberculous drugs.
【学位授予单位】：长春工业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

【引证文献】