结核分枝杆菌esat6-cfp10融合基因疫苗的构建及表达

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

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HindⅢ和EcoRⅠ双 酶切后克隆入含esat6基因的pGEM 7zf(+)载体中,将测序 正确的esat6 cfp10融合基因按照BamHⅠ和EcoRⅠ酶切位点 亚克隆入真核表达载体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基因序列与文献报道一致,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 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.

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.