【摘要】 目的 :构建结核分枝杆菌MPT6 4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MPT6 4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 -MPT6 4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MPT6 4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细胞中表达了MPT6 4。结论 :成功地构建了 pcDNA -MPT6 4质粒 ,其真核表达的蛋白具有良好的抗原性 ,为进一步研究MPT6 4在抗结核菌感染中的预防作用奠定了基础。更多还原

【Abstract】 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.更多还原