重组结核分枝杆菌Mr 38 000蛋白的表达、纯化和鉴定
重组结核分枝杆菌Mr 38 000蛋白的表达、纯化和鉴定 Cloning, Expression and Purification of Mr 38 000 protein from Mycobacterium tuberculosis ZHANG Ming， LI Jin-cheng， LIN Hang (Department of internal neurology, 2. emergency department, the Fu Zhou general hospital, Fu Zhou, 350025, P. R. China) [Abstract] To obtain Mycobacterium tuberculosis Mr 38 000 protein from H37Rv-DNA, express efficiently in E. coli and purify the Mr 38 000 proteins. Mr 38 000 protein gene was amplified by PCR from the genome of H37Rv and cloned into pGEM-T-Easy vector. After sequenced, Mr 38 000 protein gene was recombinated expression vector pQE-80L by restriction enzyme digestion, named pQE-80L-Mr 38 000. The plasmid pQE-80L-Mr 38 000 was transformed into E.coli DH5α and induced by IPTG. Mr 38 000 protein expression was analyzed by SDS-PAGE and confirmed by western blot with mice-specific mAb against (His)6. Mr 38 000 protein gene was identical with GeBank reported. The pQE-80L-Mr 38 000 vector expressed protein with relative molecule weight about 38.5 kD, which could be caught by (His)6 mAb. The expressed protein could be purified by Ni-NTA system kit with denative condition. Mr 38 000 protein gene had been successfully cloned and efficiently expressed in E.coli, The results established the basis for further study of the function of Mr 38 000 protein. [Key words] Mr38 000 protein;expression;
purification;
Mycobacterium tuberculosis (MTB) １ 材料和方法 1.1材料 MTB毒株H37Rv、 DH5α由本室保存;
pGEM-T-Easy及Wizard Plus Purification system购自Promega公司；
X-gal， 限制性内切酶BamHⅠ, EcoR Ⅰ及T4-DNA连接酶，TaqDNA聚合酶均为TaKaRa公司产品；
IPTG, DTT, DTE, 小 量质粒提取试剂盒均为Sigma公司产品，胶回收试剂盒为Vitagene公司产品。
1.2 方法 1.2.3 PCR产物的克隆与鉴定 PCR产物用琼脂糖凝胶电泳回收DNA条带。
取纯化的PCR产物与质粒pGEM-T-Easy进行连接反应，转化感受态DH5α， 均匀 涂布于含有x-gal (33μL) 和异丙基β-D硫代半乳糖苷IPTG (20μL)的LB培养皿中， 37℃培养16 h， 挑取白色菌落进行酶切鉴定，所获阳性克隆命名为pGEM-T-Easy- Mr 38 000，送至上海生工生物工程有限公司进行序列测定。1.2.6 目的蛋白的可溶性分析 大规模培养细菌，诱导表达目的蛋白，收集 细菌，超声破菌。离心后将上清和沉淀分别制样，进行SDS-PAGE分析。
1.2.7 目的蛋白的纯化 根据Ni-NTA试剂盒的说明书， 将融合蛋白与 NI-NTA结合， 用不同pH值咪唑溶液进行洗脱， 得到纯化产物。
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