结核分枝杆菌Rv1009基因蛋白的克隆和表达

目的克隆表达结核分枝杆菌促Rv1009基因,序列测定正确后进行融合、表达。方法采用热启动聚合酶链反应(Polymerase Chain Reaction,PCR)从结核分枝杆菌H37Rv基因组中扩增出Rv1009编码基因,用限制性内切酶消化后插入pGEX 4T-2载体中,将重组质粒转化大肠杆菌BL21 (DE3),目的基因经IPTG诱导,表达Rv1009基因蛋白。结果经PCR扩增在1300bp处发现一条目的片段,获得了结核分枝杆菌H37Rv株Rv1009基因蛋白,经诱导后高效表达分子量为64KD的外源蛋白,与预期分子量大小一致,凝胶自动扫描分析,在A600值为0．6,IPTG终浓度为0．3 mmol／L,诱导表达3 h时融合蛋白表达量即达峰值,占菌体总蛋白的22．8％。结论构建了结核分枝杆菌Rv1009基因重组表达载体,获得了RPF样融合蛋白的高效表达,为今后深入研究奠定了基础。

Clone and expression Of Mycobacterium tuberculosis gene Rv1009 in E. coli

Objective To clone Rv1009 gene segment from Mycobacterium tuberculosis and induce its prokaryotic expression. Methods Polymerase chain reaction ( PCR) was used to amplify the encoding gene of Rv1009 protein from Mycobacterium tuberculosis H37Rv,and the amplified DNA fragment was inserted into plasmid pGEX-4T-2. The recombinant was transformed into E. coli BL21,and then was induced for the expression of the Rv1009 fusion protein by IPTG. Results The Length of the amplified DNA fragment was 1300bp, sequence analysis showed the amplified fragment was identical With what we designed. The recombinant plasmid was successfully constructed. A 64kD fusion protein was obtained after E. coli BL21 containing recombinant plasmid was induced by 0.3 mmol/L IPTG and accounted for 22.8% of the total protein. Conclusion The colne and expression of Rv1009 were successful, which lays a basis for further study on Mycobacterium tuberculosis.