治疗性双质粒HBV DNA疫苗的体外转染表达与鉴定

Expression and Identification of Therapeutic HBV DNA Vaccine in vitro

目的:探讨治疗性双质粒HBVDNA疫苗转染COS-7细胞后目的基因的表达情况。方法:以脂质体转染试剂LipofectamineTM2000介导,将带有preS2+S基因的重组疫苗质粒pS2.S和带有hIL2+hIFNγ融合基因的重组佐剂质粒pFP分别转染COS-7细胞,同时设立空质粒转染和未转染细胞为对照组,于转染后24,48,72,96h采用ELISA法检测细胞培养上清的HBsAg、融合蛋白白细胞介素(IL-2)及干扰素(IFN-γ)的表达,并测定融合蛋白的IL-2、IFN-γ活性,采用ECLWesternblotting分析和鉴定目的蛋白preS2+S、IL-2及IFN-γ的分子质量大小。结果:双质粒在转染COS-7细胞后不同时间均可进行分泌表达,且48h时目的蛋白的表达量达峰值,其中表达的融合蛋白具有较高的IL-2和IFN-γ的生物学活性,pS2.S和pFP的转染上清分别在30.6ku和110ku处有特异性阳性反应条带。结论:HBVDNA疫苗质粒pS2.S和佐剂质粒pFP在COS-7细胞的最佳表达时间是转染后48h,并鉴定双质粒表达产物的分子质量大小分别为30.6ku和110ku。

Objective： To study the gene expression of therapeutic HBV DNA vaccine transfected into COS-7 cells, Methods： Recombinant vaccine plasmids pS2.S with preS2＋S and adjuvant plasmids pFP with hIL2＋hIFNγ were transfected into COS-7 cells by Lipofectamine recombinant TM2000 respectively. Empty plasmid and no transfected cells groups were separated. HBsAg, IL-2 and IFN-γ expressions were anayzed by ELISA at 24 h, 48 h, 72 h and 96 h after transfection. IL-2 and IFN-γexpressions were measured, and the molecular weights of preS2＋S, IL-2 and IFN-γ were analyzed and identified by ECL Western blotting. Results： Two recombinant plasmids could express in eukaryotic cells at anytime after cos-7 transfeetion, and reached the peak at 48 h after transfection. The fusion protein showed IL-2 and IFN-γ biological activity. There were single positive strips of pS2.S in 30.6 ku and pFP in 110 ku. Conclusion： The best expression time of pS2.S and pFP of HBV DNA vaccine in COS-7 cell is 48 h after transfeetion. The molecular weights of the two products are 30.6 ku and 110 ku respectively.