结核分枝杆菌和卡介苗菌株中喹啉酸磷酸核糖转移酶(QPRT)的原核表达及酶活性测定  

Procaryotic expression and activity determination of quinolinic acid phosphoribosyl transferase from Mycobacterium tuberculosis H37Ra and Mycobacterium bovis bacilli-Calmette-Guerin str.Pasteur 1173P2

作　　者：葛赛 孙曼銮 GE Sai;SUN Manuan(Center of Academic Journal,Shanxi Datong University,Datong 037009,China;Institute of Carbon Materials,Shanxi Datong University,Datong 037009,China;School of Medicine,Shanxi Datong University,Datong 037009,China)

机构地区：[1]山西大同大学学术期刊中心,山西大同037009 [2]山西大同大学炭材料研究所,山西大同037009 [3]山西大同大学医学院,山西大同037009

出　　处：《黑龙江畜牧兽医》2023年第21期31-36,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：山西省基础研究计划项目(202103021223339,20210302124435);山西省回国留学人员科研项目(2022-175);山西省高等学校科技创新项目(2019L0773,2019L0771);山西大同大学博士启动金项目(2018-B28,2018-B13)。

摘　　要：为了在原核表达系统E.coli中表达并纯化结核分枝杆菌(Mycobacterium tuberculosis,M.tuberculosis)H37Ra和卡介苗菌株(Mycobacterium bovis bacilli-Calmette-Guerin,BCG)str.Pasteur 1173P2的喹啉酸磷酸核糖转移酶(quinolinic acid phosphoribosyl transferase,QPRT),探索不同来源QPRT蛋白结构,并测定其酶活性,试验采用PCR方法分别对M.tuberculosis和BCG中QPRT的编码基因nadC进行扩增,与pET-28a载体连接构建重组载体并在E.coli中完成原核蛋白的表达,在体外进行目的蛋白的纯化,利用高效液相色谱法检测不同来源的QPRT蛋白酶活性,使用PyMOL v2.3.2软件分析其氨基酸残基结构。结果表明:在体外成功克隆了M.tuberculosis和BCG中的大小为858 bp的编码基因nadC,并成功构建出重组载体pET-28a-nadC-MTB和pET-28a-nadC-BCG,在E.coli中进行QPRT蛋白的表达与体外纯化后获得分子量为34 ku的QPRT蛋白;两种不同菌株来源的QPRT蛋白以喹啉酸(quinolinic acid,QA)为底物时酶活性无差异;不同来源的QPRT蛋白中存在1个差异氨基酸残基位点,但酶活性并无明显差异。说明QA的结构类似物吡嗪酸(pyrazinoic acid,POA)不是QPRT的底物,为抗结核药物靶点的探索提供了潜在价值。In order to express and purify Mycobacterium tuberculosis in E.coli,a prokaryotic expression system,M.tuberculosis H37Ra and Mycobacterium bovis bacilli-Calmette-Guerin(BCG)str.Pasteur 1173P2 quinolinic acid phosphoribosyl transferase(QPRT),and to explore the structure of QPRT proteins from different sources and determine their enzyme activity,PCR was used to amplify the QPRT encoding gene nadC from M.tuberculosis and BCG,and the recombinant vector was connected to pET-28a vector,and the prokaryotic protein was expressed in E.coli,and the target protein was purified in vitro.The activity of QPRT protease from dfferent sources was detected by HPLC,and the structure of amino acid residues was analyzed by PyMOL v2.3.2 software.The results show that the 858 bp nadC gene of M.tuberculosis and BCG was successfully cloned in vitro,and the recombinant vectors pET-28a-nadC-MTB and pET-28a-nadC-BCG were successfully constructed.QPRT protein was expressed in E.coli and purified in vitro,and the molecular weight of QPRT was 34 ku.There was no difference in enzyme activity of QPRT protein from two different strains when quinolinic acid(QA)was used as substrate.There was a difference of one amino acid residue in QPRT protein from different sources,but there was no significant difference in enzyme activity analysis.These results indicated that pyrazinoic acid(POA),a structural analogue of QA,was not a substrate of QPRT,which provided potential value for the exploration of anti-tuberculosis drug targets.

关 键 词：结核分枝杆菌 卡介苗 喹啉酸磷酸核糖转移酶 蛋白表达 酶活性测定 

分 类 号：S852.618[农业科学—基础兽医学] Q936[农业科学—兽医学]