牛分枝杆菌mpb64-ag85b-esat-6融合基因真核表达载体的构建及其在SP2/0细胞中的表达

目的构建牛分枝杆菌mpb64-ag85b-esat-6融合基因真核表达载体,并在SP2/0细胞中表达。方法利用PCR技术和重叠延伸剪接(SOE)技术扩增牛分枝杆菌ag85b、mpb64、esat-6基因和mpb64-ag85b融合基因,克隆至真核表达载体pcDNA3.1(+)上,构建重组质粒pcMAE。将该重组质粒体外转染SP2/0细胞,间接免疫荧光法检测目的基因的表达。结果真核表达质粒pcMAE经酶切鉴定及测序,证实构建正确,转染后SP2/0细胞膜上出现均质的蓝绿色荧光。结论已成功构建了牛分枝杆菌mpb64-ag85b-esat-6融合基因真核表达载体,并在SP2/0细胞中获得表达,为进一步研制牛结核病多基因融合DNA疫苗奠定了基础。

Construction of Eukaryotic Expression Vector for mpb64-ag85b-esat-6 Fusion Gene of Mycobacterium bovis and Its Expression in SP2/0 Cells

Objective To construct the eukaryotic expression vector for mpb64-ag85b-esat-6 fusion gene of Mycobacterium bovis and express in SP2/0 cells. Methods Amplify ag85b, mpb64 and esat-6 genes from genome of M. bovis strain Vallee Ⅲ by PCR respectively. Splice the amplified mpb64 and ag85b genes by overlapping extension (SOE) technique to obtain mpb64-ag85b fusion gene. Insert mpb64-ag85b fusion gene and esat-6 gene into eukaryotic expression plasmid pcDNA3.1(+), and transfect SP2/0 cells with the constructed recombinant plasmid pcMAE in vitro. Identify the expression of target protein by indirect immunofluorescence test. Results Both restriction analysis and sequencing proved that recombinant plasmid pcMAE was constructed correctly. Homogeneous bluish-green fluorescence was observed on the membrane of SP2/0 cells transfected with pcMAE. Conclusion The eukaryotic expression vector for mpb64-ag85b-esat-6 fusion gene of M. bovis was successfully constructed and expressed in SP2/0 cells, which laid a foundation of multiple gene fusion DNA vaccine for prevention of bovine tuberculosis.