CD/TK单、双自杀基因重组腺病毒的制备及初步应用

Construction of Recombinant Adenoviruses Expressing CD/TK Suicide Genes and Its Preliminary Application

为制备表达大肠杆菌胞嘧啶脱氨酶 (cytosinedeaminase ,CD)和单纯疱疹病毒 1 胸苷激酶(herpessimplexvirustype 1thymidinekinase ,TK)双自杀基因的重组腺病毒载体 ,为再狭窄基因治疗的应用奠定基础 ,首先构建了含单、双自杀基因的腺病毒穿梭载体 ,细菌内同源重组法获得重组腺病毒质粒 .脂质体介导下转染 2 93细胞进行包装并大量扩增得到Ad TK、Ad CD、Ad TK CD、Ad LacZ 4种重组腺病毒 .氯化铯 (CsCl)梯度离心法纯化后利用报告基因绿色荧光蛋白 (GFP)测定病毒滴度 ,可达 10 9pfu ml .PCR和Western印迹法对外源基因进行鉴定 ,证明单、双自杀基因均已整合入腺病毒基因组并表达 .重组腺病毒感染血管平滑肌细胞后 ,用MTT法比较了单、双自杀基因的杀伤作用 ,发现在感染复数≤ 2 0时 ,双自杀基因对细胞的抑制作用明显高于单基因 .成功地构建腺病毒双自杀基因共表达载体 ,为进一步的体内外实验奠定了基础 .

In order to prepare recombinant adenoviruses expressing CD/TK suicide genes to prevent restenosis, shuttle adenoviral vectors containing CD and/or TK genes were constructed. 4 recombinant adenovirus constructs, namely Ad TK, Ad CD, Ad TK CD and Ad lacZ were obtained by homologous recombination in recA+ E.coli cells. The adenoviruses were amplified in 293 package cells and purified in a CsCl gradient. The titers could reach 10 9 pfu/ml by GFP detection. The expression of the single and double genes was confirmed by PCR and Western blot analysis. The vascular smooth muscle cells (VSMCs) were infected by recombinant adenovirus expressing TK and/or CD. Their antiproliferation effect on VSMCs was detected by MTT assay. It was showed that the infected VSMCs with double gene had greater inhibition effect than both the Ad CD and Ad TK groups when the multiplicity of infection ( moi )≤20. The results demonstrated that recombinant adenovirus expressing single or double suicide genes were successfully obtained, it could be used for further investigation of in vivo and in vitro experiments.