结核分枝杆菌esat6-cfp10融合基因疫苗的构建及表达

目的：构建结核分枝杆菌esat6-cfp10融合基因疫苗并对其在体外进行表达。方法：用PCR技术从MTB毒株H37Rv基因组DNA中扩增cfp10基因，以Hindm和EcoR Ⅰ双酶切后克隆人含esat6基因的pGEM-7zf( )载体中，将测序正确的esat6-cfp10融合基因按照BamH Ⅰ和EcoR Ⅰ酶切位点亚克隆人真核表达载体pcDNA3．1( )，重组质粒酶切鉴定正确后以脂质体转染CHO细胞，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．结果：PCR获得的cfp10基因序列与献报道一致，大小约为350bp．重组真核表达质粒酶切后可获得约630bp的融合esat6-cfp10基因片段．RT—PCR可获得大小约为350bp的诉CFP10基因，间接免疫荧光检测后表达有Esat6-Cfp10蛋白的阳性细胞着染．结论：成功克隆结核分枝杆菌CFP10基因，构建了融合有esat6-cfp10基因的真核表达质粒并对其在体外进行了表达。

Construction and expression of fused gene vaccine esat6-cfp10 of Mycobacterium tuberculosis

AIM: To construct and express fused gene vaccine esat6-cfp10 of Mycobacterium tuberculosis. METHODS: cfp10 gene was amplified by PCR from H37Rv virulent strain of MTB and was then inserted into pGEM-7zf(+) cloning vector containing esat6 gene. After sequenced, the esat6-cfp10 fused gene was cloned into eukaryotic vector pcDNA3.1(+). This recombinant plasmid was transfected into Chinese Hamster Ovary (CHO) cells by cation liposome. The expression of mRNA and the fused protein were detected by RT-PCR and indirect immunofluorescence technique. RESULTS: The length of PCR product was 350 bp, identical with that reported by GenBank. The 630 bp fragment of the fused esat6-cfp10 gene was obtained by restriction enzyme digestion. RT-PCR suggested that the mRNA was expressed in CHO cells and the positive cells were stained positively by indirect immunofluorescence technique. CONCLUSION: cfp10 gene was successfully cloned and the eukaryotic recombinant plasmid fused esat6-cfp10 was constructed successfully and expressed in CHO cells.