gp350及gp350-C3d3融合基因真核载体的构建及其免疫功能研究

Construction of gp350 gene and gp350-C3d3 fusion gene eukaryotic expression vectors and the study of its immunity

目的构建EB病毒包膜糖蛋白gp350全长序列及其与3个补体片段C3d串联基因在人体真核细胞中表达的载体,并进行其免疫功能的初步研究。方法将gp350基因序列分别与经BglⅡ和BamHⅠ双酶切的真核表达载体pSG.SS.YL和pSG.SS.C3d3.YL连接,转化后挑取阳性克隆进行PCR、酶切鉴定并测序;将重组载体利用脂质体法分别转染人类Hela细胞培养16~24 h后,用Western blot及细胞免疫组化染色检测gp350蛋白和gp350-C3d3融合蛋白的表达。结果成功构建含gp350全长基因序列的载体pSG.SS.gp350.YL和pSG.SS.gp350.C3d3.YL,并能够转染人类Hela细胞;经细胞免疫组织化学染色及Westernblot蛋白检测转染组均能常规水平表达,对照组则无该蛋白表达,但两转染组的表达量无明显差异。结论构建成功含有gp350的2个真核表达质粒并能够在人体细胞中表达相应蛋白,为下一步进行检测功能性试验奠定基础。

To study the function of gp350,we constructed two eukaryotic expression vectors containing gp350 gene.gp350 full-length cDNA was amplified by polymerase chain reaction with plasmid of pcDNA3.1＋gp350 as a template.PCR product was digested by restriction enzymes BglⅡand BamH,and then cloned to sites between BglⅡand BamHⅠof pSG.SS.YL and pSG.SS.C3d3.YL,respectively.The positive clones were identified by PCR,restriction enzymes digestion and DNA sequencing.Hela cells were transient transfected by recombinant plasmids and the expression of gp350-C3d3 fusion protein was detected by immunohistochemistry and Western blot.The results above indicate that the recombinant gp350-C3d3 fusion gene is successfully constructed,which could be a foundation for further research on the immunity of gp350 against EBV virus.