结核分枝杆菌酯酶Rv1400c原核表达及表达产物的酶活性分析  

Prokaryotic Expression of Esterase Rv1400c from Mycobacterium tuberculosis and Determination of Expressed Product Enzyme Activity

作　　者：陶荣珊[1,2] 李金伟 刘思国[4] 王全凯[1,2] 

机构地区：[1]吉林农业大学动物科学技术学院,长春130118 [2]吉林省中韩动物科学研究院,长春130600 [3]吉林东丰药业股份有限公司,东丰136300 [4]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/动物细菌病研究室,哈尔滨150001

出　　处：《吉林农业大学学报》2014年第6期701-706,共6页Journal of Jilin Agricultural University

基　　金：国家国际科技合作专项(2011DFA32900);国家自然科学基金项目(31100659;31272538);国家重点基础研究发展计划项目[973计划(2012CB518801)];长春市科技局项目[长科技合(2011239)]

摘　　要：为探究结核分枝杆菌酯酶Rv1400c活性，以进一步研究Rv1400c的结构和功能。以结核分枝杆菌H37Rv基因组DNA为模板，对 Rv1400c 基因进行扩增，并将其克隆到 pET28b （＋）原核表达载体上，构建pET28b－Rv1400c重组质粒，然后将构建的重组质粒转化到BL21（ DE3）表达菌株中，增菌后用IPTG进行诱导表达再用亲和层吸树脂法进行纯化，利用不同底物、不同pH、不同温度对纯化后的Rv1400c酯酶进行活性分析。结果表明：成功构建出pET28b－Rv1400c重组质粒；SDS－PAGE和Western blot显示，Rv1400c以包涵体形式表达，蛋白分子质量为39 ku；在对8种底物的筛选中发现Rv1400c在C2～C14中具有活性，其中以在C2中活性最好；在以C12为底物、pH 8？0、37℃条件下酯酶活性最高。This study aims to explore the activity of esterase Rv1400c of Mycobacterium tuberculosis and do further researches on the structure and function of Rv1400c. The Rv1400c gene was amplified with PCR and cloned into pET-28b（＋） prokaryotic expression vector by using Mycobacterium tuberculosis H37Rv genomic DNA as template. Recombinant plasmid of pET28b-Rv1400c was constructed and then translated into BL21 （ DE3 ） expression strain. It was induced to express with IPTG after bacterial splitting and then purified with Ni-NTA affinity chromatography method. Activi ty of the purified Rv1400c was analyzed with different substrates, pH and temperature of three directions. The results showed that recombinant plasmid of pET28b-Rv1400c was successfully constructed;SDS-PAGE and Western blot showed Rv1400c was expressed in inclusion bodies and molecular weight of the protein was 39 ku;The Rv1400c was found active within the range of C2 to C14 in the screening of eight kinds of substrates and the best activity was in C2;Esterase activity was the highest under the conditions of pH 80 and 37 ℃ when the C12 was the substrate.

关 键 词：结核分枝杆菌酯酶Rv1400c pET28b-Rv1400c重组质粒 包涵体 酯酶活性 

分 类 号：S852.61[农业科学—基础兽医学]