摘要
目的 :克隆结核分枝杆菌 (Mtb)Rv2 4 5 0基因 ,序列测定正确后进行融合表达、纯化 .方法 :采用聚合酶链反应(PCR)从MtbH37Rv基因组中扩增出Rv2 4 5 0编码基因 ,序列测定正确后 ,亚克隆到融合表达载体 pPro EXHT中 ,转化大肠杆菌DH5α ,目的基因经IPTG诱导 ,由T7启动子调控表达带 6个组氨酸残基的Rv2 4 5 0蛋白 ,在变性条件下对目的蛋白进行亲和层析纯化 .结果 :得到融合 6个组氨酸残基的Rv2 4 5 0蛋白纯度大于 90 % .结论 :构建了MtbRv2 4 5 0基因的重组表达载体 ,并获得了高纯度的融合表达蛋白 .
AIM: To obtain Rv2450 gene segments of Mycobacterium tuberculosis (Mtb), express them efficiently in E.coli and purify the aim proteins. METHODS: Rv2450 gene segments were amplified by PCR with specific primers from genome of Mtb H37Rv strain. After sequenced, Rv2450 gene segments were subcloned into expression vector pPro-EX HT and purified in E.coli DH5α. Recombinant 6×His fused proteins were purified via Ni-NTA purification system. RESULTS: The length of PCR products was 529 bp and identical with what the GenBank reported. SDS-PAGE analysis showed that the fusion protein mainly existed in inclusion bodies. We isolated the inclusion bodies from the culture and purified the fusion protein under denaturing condition using metal chelate affinity chromatography. CONCLUSION: The construction of the recombinant expressing plasmid lays a basis for further study of Rv2450 protein.
出处
《第四军医大学学报》
北大核心
2004年第19期1778-1781,共4页
Journal of the Fourth Military Medical University
基金
军队"十五"重点项目 (0 1z0 83)
关键词
结核分枝杆菌
克隆
表达
纯化
Mycobacterium tuberculosis
cloning
expression
purification
