结核分枝杆菌分泌蛋白MPT64的克隆、表达与鉴定

Cloning and expression of secreted protein MPT64 of Mycobacterium tuberculosis

目的 :构建表达结核分枝杆菌分泌蛋白MPT6 4重组质粒 ,并在大肠杆菌中表达获得基因重组蛋白 .方法 :用PCR方法从结核分枝杆菌H37Rv基因组扩增出MPT6 4基因片段 ,插入 pGEM T easy载体中 ,序列测定正确后 ,将其亚克隆到表达载体pProEXHTb中 ,在大肠杆菌DH5α中表达 .结果 :获得结核分枝杆菌MPT6 4成熟蛋白基因 ,经序列测定与GenBank公布的序列完全一致 ;表达蛋白经SDS PAGE分析 ,相对分子质量与文献报道一致 ;重组蛋白经蛋白印迹实验 ,在Mr2 .6× 10 4位置有特异条带 .MPT6 4蛋白在宿主菌中表达量约占菌体总蛋白的 13.5 % .结论 :基因重组表达的融合蛋白有可能作为有效抗原用于结核病的预防疫苗的研制和皮肤免疫诊断试剂的制备 .

AIM: To construct the recombinant plasmid of secreted protein MPT64 of mycobacterium tuberculosis and to express fusion protein in E.coli DH5α. METHODS: The gene encoding MPT64 protein was amplified by polymerase chain reaction (PCR) from genome of Mycobacterium tuberculosis H37Rv strain and inserted into cloning vector pGEM T easy after restriction endonuclease digestion. After the sequencing was confirmed, the gene was subcloned to the expression vector pProEX HTb. Recombinant MPT64 was expressed in E.coli DH5α. RESULTS: The DNA sequence of mpt64 was identical with that published by GenBank. The relative molecular mass ( M r) of the protein corresponded to the Mr shown in SDS PAGE. The express protein was confirmed by Western blot analysis. CONCLUSION: The recombinant protein may be a potential candidate to be used as a vaccine or skin test reagent.