重组结核分枝杆菌Mr 38 000蛋白表达、纯化及鉴定.docVIP

重组结核分枝杆菌Mr 38 000蛋白表达、纯化及鉴定 　摘要 从H37Rv基因组中扩增Mr38 000 蛋白基因并高效表达和纯化。用PCR技术从结核分枝杆菌H37Rv基因组中扩增Mr 38 000蛋白序列，将其与pGEM-T-Easy 载体连接转化E.coli DH5α，构建重组克隆载体pGEM-T-Easy/ Mr 38 000，测序正确后将目的基因片断克隆入pQE-80L原核表达载体并转化E.coli DH5α，IPTG诱导目的蛋白表达。 经Western-blot 鉴定目的基因与(His)6融合表达，将已表达的蛋白质通过Ni-NTA亲和色谱柱进行纯化。PCR得到结核分枝杆菌Mr 38 000蛋白基因，测序结果与GeBank 中报道的完全一致。SDS显示， 在Mr为38.5×103处有相应的蛋白质表达条带，Wesern blot鉴定为(His)6融合表达蛋白。经Ni-NTA亲和色谱柱进行纯化后可得到纯化的蛋白。成功克隆了铁调节蛋白基因Mr38 000 蛋白序列，并在E.coli DH5α高效表达，亲和层析后获得了纯化目的蛋白。 关键词 Mr 38 000蛋白; 表达; 纯化; 结核分枝杆菌； 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 SDSand 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) 结核分枝杆菌（Mycobacterium tuberculosis, MTB）是结核病(tuberculosis, TB)的病原体。其在患者体内主要为细胞内生长。诊断方法的欠缺是结核病流行的重要原因。 目前常用的PPD