p27kip1复制缺陷腺病毒重组体的构建与鉴定

目的 :构建含人细胞周期依赖激酶抑制剂p2 7kip1基因的重组腺病毒载体 ,为采用p2 7kip1cDNA防治肿瘤的研究奠定基础。方法 :将人p2 7kip1cDNA插入到穿梭质粒pACCMVPLPA的CMV启动子之下 ,即pAd p2 7kip1。后者与pJM17通过脂质体共转染 2 93细胞 ,经同源重组获得含人p2 7kip1cDNA的重组腺病毒Ad p2 7kip1。结果 :p2 7kip1cDNA成功地插入了pACCMVPLPA载体 ,以重组病毒基因组DNA为模板 ,同时扩增出了 2 75bp的p2 7kip1基因片段和 86 0bp的腺病毒骨架基因片段 ,证实了Ad p2 7kip1的正确性。病毒滴度为 1.2 4× 10 1 2 pfu ml。结论 :成功构建了携带人p2 7kip1基因的腺病毒Ad p2 7kip1,为采用p2 7kip1cDNA途径防治消化道肿瘤的在体、离体实验奠定了基础

Construction and Assessment of Replication-Deficient Adenoviral Vector Containing the cDNA for Human p27kip1

Objective: To construct the recombinant adenovirus vector containing the cDNA for p27kip1 for future study of tumor treatment. Methods: The p27kip1cDNA had been extracted from pCMV5p27kip1 with KpnI and BamHI,and then inserted into the E1 deleted expression plasmid pACCMVPLPA shuttle vector,called pAd p27kip1. pAd p27kip1 was cotransfected with the plasmid pJM17 into the transformed human embryonic kidney cell line 293 cells by liposome mediated method. Homologous recombination of the pAd p27kip1 and pJM17 in 293 cells replaced the E1 regine with the expression cassette from pAd p27kip1. Ad p27kip1 was confirmed by PCR.Ad p27kip1 was propagated in 293 cells and then underwent CsCl density purification. Subsequently, the preparations were didalyzed in dialysis buffer. The titer of each viral stock was determined by measuring the absorbance at 260 nm. Results: p27kip1cDNA was successfully inserted into the shuttle vector pACCMVPLPA. pAd p27kip1 was confirmed by Kpn1 and BamH1 digestion. Ad p27kip1 was characterized by PCR coamplification. The virus titer was 1.24×10 12 pfu/ml. Conclusion: Ad p27kip1 containing the p27kip1 sequence was successfully constructed. This investigation provides the basis for future study of tumor treatment based on p27kip1cDNA strategy.