以AdMax载体系统构建和制备肾癌相关抗原G250基因重组腺病毒表达载体

目的 构建肾癌相关抗原G250重组腺病毒载体,以期用于基因转染树突状细胞(DC)治疗肾癌的实验研究.方法 利用AdMax包装系统,首先构建穿梭质粒pDC316-G250,然后将所得的穿梭质粒和辅助质粒pBHGlox(delta)E1,3Cre以CaCL2法质粒共转染293细胞得到重组腺病毒Ad/G250,CsCl梯度离心纯化病毒,TCID50法测定病毒滴度.Ad/G250转染DC细胞,RT-PCR及流式细胞仪检测G250的表达.结果 采用双质粒共转染293细胞,Cre/loxP位点同源重组方法构建了E1和E3缺失的含外源基因的Ad/G250载体.经过PCR、RT-PCR和流式细胞仪证实腺病毒载体构建成功.病毒经过CsCl梯度离心纯化后,Ad/G250滴度达到5.6×109 U/ml.RT-PCR及流式细胞仪显示Ad/G250在DC中特异性表达.结论 成功构建了G250重组腺病毒载体.

Construction of a recombinant adenovirus expression vector for human renal tumor-associated antigen G250 gene with AdMax system

OBJECTIVE: To construct a adevoviral vector harboring human renal tumor-associated antigen G250 gene for transfecting the dendritic cells (DCs) and treating renal tumors. METHODS: The G250 genes were cloned into the shuttle plasmid pDC316 to construct pDC316-G250, which was cotransfected with the rescue plasmid pBHGlox(delta)E1,3Cre into 293 cells to obtain the recombinant adenovirus Ad/G250. The inserted gene and its expression were identified by RT-PCR and fluorescence-activated cell sorting (FACS) after recombinant adenovirus transfection of the DCs. The recombinant adenoviral vector was purified by CsCl banding and titrated by TCID50. RESULTS AND CONCLUSION: The recombinant adenoviral vector of G250 gene was successfully constructed and high titer of the recombinant adenoviruse was obtained. G250 mRNA and protein expressions were identified in Ad/G250-transfected DCs. The titer of the virus stocks reached 5.6x10(9) IU/ml.