结核分枝杆菌MPT64真核表达质粒的构建及其表达

目的：构建结核分枝杆菌MPT64真核表达质粒，并在真核细胞中表达。方法：从H37Rv基因组中扩增出MPT64基因，经限制性内切酶消化后，定向插入pcDNA3.1( ）中，用脂质体法将pcDNA-MPT64转染COS-7细胞。采用RT-PCR、ELISA和斑点印迹法检测其表达。结果：扩增出的MPT64基因正确插入pcDNA3.1中，DNA序列测定无突变产生。重组质粒在COS-7细胞中表达MPT64。结论：成功地构建了pcDNA-MPT64质粒，其真核表达的蛋白具有良好的抗原性，为进一步研究MPT64在抗结核菌感染中的预防作用奠定了基础。

Construction and expression of the eukaryotic expression plasmid encoding mycobacterium tuberculosis MPT64

Objective:To construct the recombinant eukaryotic plasmid encoding Mycobacterium tuberculosis MPT64 and detect its expression in COS-7 cell lines. Methods: The gene encoding MPT64 was amplified by polymerase chain reaction (PCR) from H37Rv genome ,and the fragment was inserted into pcDNA3.1(+) after restriction endonuclease digestion .The recombinant plasmid pcDNA MPT64 was transfected into COS-7 cell lines with liposome ,and expressed MPT64 in COS-7 cell was detected by RT-PCR,ELISA and dot blotting. Results: The sequence of MPT64 gene in recombinant plasmid was the same as that of reported,and recombinant MPT64 protein was expressed in COS-7 cell lines.Conclusion: The recombinant plasmid pcDNA MPT64 was constructed successfully,and MPT64 protein expressed in COS-7 cell has antigenicity.The results established the basis for further studies on MPT64 function.