结核分枝杆菌mpt64基因真核表达载体的构建及表达

构建结核分枝杆菌分泌蛋白mpt64基因真核表达载体。通过PCR法从MTBH37Rv株基因组中扩增mpt64基因，插入pGEM-T-easy载体中，序列测定正确后，将其亚克隆到真核表达载体pcDNA3．1（-），重组质粒酶切鉴定正确后，以Lipo-fectamine2000转染COS-7细胞后，分别以RT-PCR方法检测mRNA表达和间接免疫荧光技术检测目的蛋白的表达。构建了真核表达载体pcDNA-mpt64，RT-PCR结果证明mpt64基因可在COS-7细胞中转录，用间接免疫荧光检测，有表达mpt64蛋白的细胞着染。结果表明，构建结核分枝杆菌mpt64基因的真核表达载体pcDNA-mpt64成功，mpt64基因可以在COS-7细胞中表达。

Construction of the Fused Eukaryotic Expression Vector of Mycobacterium tuberculosis mpt64 Gene and Its Expression

In order to construction the fused eukaryotic expression vector of Mycobacterium tuberculosis mpt64 gene and express it in COS-7 cell.The gene encoding mpt64 protein was amplified by polymerase chain reaction(PCR)from genome of Mycobacterium tuberculosis H37Rv strain,and first inserted into cloning vector pGEM-T-easy.After sequencing was confirmed,the gene was subcloned to eukaryotic expression vector pCDNA3.1(-).The recombinant plasmid pCDNA-mpt64 was transfected into COS-7 cells with liposome.The expression was detected by RT-PCR and indirect immunofluorescence.Eukaryotic recombinant plasmid encoding mpt64 were constructed successfully.The mpt64 gene can be expressed in COS-7 cell.