摘要
目的:克隆并原核表达结核分枝杆菌Rv2389c基因。方法:培养结核分枝杆菌,提取其基因组,PCR扩增出485bp的Rv2389c基因,克隆入pSP73载体,测序分析。将该目的基因亚克隆入pGEX-4T-2表达载体,测序证实正确后转化大肠杆菌BL21(DE3),待大肠杆菌增菌至A600nm值为0.6时,用0.3 mmol/L IPTG在30℃诱导表达重组蛋白。结果:测序结果证实获得了Rv2389c基因,其序列与GenBank中报道完全一致。构建了重组表达质粒,表达的蛋白以12%SDS-PAGE分析,在Mr约42KD处出现一条新生蛋白带,经凝胶薄层扫描检测,表达量约占菌体蛋白的26.8%。结论:成功克隆了结核分枝杆菌Rv2389c基因并得到了其大肠杆菌表达产物,为进一步研究其活性和功能奠定了基础。
Objective： To clone and express mycobacterium tuberculosis Rv2389c gene. Methods： The genome NDA was extracted from the cultured mycobacterium tuberculosis Rv2389c gene was obtained by PCR using specific primers and cloned into BamHI and EcoRI site of pGEX - 4T- 2 expression vector and confirmed by sequencing. After transforming E. coli BL21 （DE3）, recombinant bacteria was induced at 30℃ for 4 to 5 h, the fusion protein GST - Rv2389c was analyzed by SDS-PAGE. Results： DNA sequencing results showed that the amplified Rv2389c gene was exactly consistent with the sequence reported in Genebank, and the SDS - PAGE analysis demonstrated that the Rv2389c was expressed in E. coli BL21 （DE3）. The protein band amounted to 26. 8% of total bacteria protein. Conclusion： Mycobaeterium tuberculosis Rv2389c gene has been cloned and expressed successfully in E.coli 21 （DE3）.
出处
《西南国防医药》
CAS
2006年第1期25-27,共3页
Medical Journal of National Defending Forces in Southwest China
关键词
结核分枝杆菌
促进复活因子
克隆
表达
mycobaeterium tuberculosis, resuscitation- promoting factor, clone, expression, gene
