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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。
OBJECTIVE: To construct the full-length gp350 sequence of Epstein-Barr virus envelope glycoprotein (gp350) and its expression vector with 3 complement C3d tandem genes in human eukaryotic cells and to study its immunological function. Methods The gp350 gene sequence was ligated with the eukaryotic expression vectors pSG.SS.YL and pSG.SS.C3d3.YL which were double digested with BglⅡ and BamHⅠ, respectively. After transformation, the positive clones were picked out for PCR, restriction enzyme digestion and sequencing; The recombinant vector was transfected into human Hela cells by lipofectamine for 16-24 h, then the expression of gp350 protein and gp350-C3d3 fusion protein was detected by Western blot and immunohistochemistry. Results The vectors pSG.SS.gp350.YL and pSG.SS.gp350.C3d3.YL containing the full-length gp350 gene were successfully constructed and transfected into human Hela cells. The transfection group was detected by immunohistochemistry and western blot Both of them could be routinely expressed, while the control group did not express the protein, but there was no significant difference between the two transfection groups. Conclusion The construction of two eukaryotic expression plasmids containing gp350 and their ability to express the corresponding proteins in human cells lay the foundation for the next functional test.