胰腺癌MUC4抗原多表位嵌合基因重组腺病毒载体的构建及其在COS-7细胞的表达

Construction of recombinant adenovirus containing pancreatic tumor associated MUC4 antigen derived polyepitope combined PARDE and expression in COS-7 cells

目的:构建含有胰腺癌MUC4抗原多表位嵌合基因的腺病毒载体,并转染真核细胞COS-7。方法:多参数分析预测MUC4抗原HLA-A1、HLA-A2限制性CD8+T细胞表位kozac序列、引导序列以及通用Th表位PARDE和多表位基因串连后合成嵌合全基因。嵌合基因克隆到腺病毒穿梭载体pAdshuttle-CMV获得重组质粒pAdshuttle-CMV-PE。酶切鉴定后,将正确的重组质粒转化含有腺病毒骨架载体的BJ5183菌进行同源重组。PacⅠ酶切和测序鉴定正确的重组子,脂质体介导转染Ad293细胞,通过观测绿色荧光蛋白(GFP)的表达和PCR扩增目的基因的方法鉴定重组腺病毒(rAd-PE)。Ad293细胞反复感染冻融扩增病毒,流式细胞仪和TCID50检测病毒滴度。病毒颗粒感染COS-7细胞,荧光显微镜观测绿色荧光蛋白的表达以及SDS-Page分析多表位嵌合蛋白的表达。结果:成功构建了含有胰腺癌MUC4的多表位嵌合基因重组腺病毒,病毒滴度为1×1010pfu/ml。SDS-Page发现在16kD处有一符合预期大小的蛋白表达。结论:该重组腺病毒成功构建并能够在真核细胞COS-7中表达,为下一步胰腺癌的肿瘤疫苗研究奠定相应的基础。

objective：To construct the recombinant adenovirus containing pancreatic tumor associated MUC4 antigen and provide the basis for further research on pancreatic tumor vaccine. Methods：The HLA-A1 and HLA-A2 restricted epitopes of MUC4 was predicted by BIMAS and ProPred. The combined polyepitope gene with kozac sequence,sign peptide and PARDE was synthesized. The combined polyepitope was subcloned into pAdshuttle-CMV shuttle plasmid （pAdshuttle-CMV-PE）,and then transformed into competent E.coli BJ5183 cells with backbone plasmid already. The homologous recombinant adenoviral plasmid pAdeasy-l-PE was validating with Pac I digesting and sequencing to verify their fidelity. Ad293 cells were transfected with Pac I linearized recombinant and lipofectamine reagent. Identified by green fluorescence protein （GFP） expression and PCR method,the virus was amplified by infection 293A cells repeatly. Viral particle concentration was determined by TCID50 plus flow cytometer. The virus was transduced into COS-7 cells,and expression of polyepitope protein was demonstrated by the GFP expression and SDS-Page analysis. Results：The recombined adenovirus containing combined polyepitope was constructed successfully and the titer was 10 pfu/ml. Conclusion：A recombinant adenovirus containing combined polyepitope was constructed successfully,a specific protein corresponding with the predicted mass was found in virus transduced COS-7 cells.