构建含有小鼠CD40-IgG2aFc-IRS-GFP融合基因腺病毒并体外检测

目的 构建载有小鼠CD40-IgG2aFc-IRS-GFP融合基因片段的腺病毒载体,并检测其转染293细胞后的融合蛋白表达及出毒情况.方法 提取小鼠CD40、IgG2aFc基因片段构建PDC316-IgG2aFc-GFP质粒,测序成功后包装构建Ad5-PDC316-CD40-IgG2aFc-GFP,转染293细胞.出毒后大量复制病毒并纯化,测定病毒滴度,体外感染细胞观察病毒复制及分泌目的 蛋白情况并检测目的 蛋白表达量.结果 成功构建了质粒及病毒,体外感染显示该病毒能有效的感染细胞并表达蛋白(荧光显示),检测目的 蛋白表达量随时间点及浓度增加而增加.当病毒浓度增加到一定程度时目的 蛋白表达趋于无明显差异性.结论 构建小鼠融合基因片段并成功转入细胞内,分泌表达目的 蛋白是可行的,为进一步研究该病毒在大鼠体内感染及表达CD40Ig、大鼠肝移植模型免疫耐受及其机制的研究奠定了基础.

Adenovirns construction with miceCD40- IgG2aFc-IRS-GFP fusion gene and in vitro testing

Objective To construct the adenovirus vector containing mice CD40- IgG2aFc-IRS-GFP fusion gene and detect the expression of the fusion protein in transfeete 293cells.Methods The fragments of CD40, IgG2aFc were obtained and inserted into plasmid PDC316-IgG2aFc-GFP.After being verified by sequencing, Ad5-PDC316-CD40-IgG2aFc-GFP was contransfored with the bckbone vector into 293 cells.The virus titer was detected after replicating and purifing and the expression of the fusion protein was analyzed.Results A virus and plasmid were constructed successfully.The vitro infection showed that the virus can infect cells of which the fusion protein was confirmed by fluo-rescence.The expression of the fusion protein increased with the increased time and virus concentra-tion.The protein expression stopped increasing after the virus concentration came to a certain level.Conclusion CD40, IgG2aFc fragments are correctly ligated with plasmid PDC316-IgG2aFc-GFP, and the fusion protein can be expressed in 293cells.This might lay a foundation for further studies of the expresstion of virus, immune tolerance and mechanism of liver transplantation model in rats.