重组pAd/CMV/V5-DEST-p16载体的构建及其对人肝癌细胞的抑制作用

Construction of recombinant p16 adenovirus vector and its suppressive role in the growth of human heptocellular carcinoma cell line

目的构建pAd/CMV/V5-DEST-p16重组腺病毒载体,并观察野生型p16基因对人肝癌细胞SMMC7721株的抑制作用。方法合成人p16-INK4表达基因。构建pENTR 1A-p16质粒。通过LR反应,入口克隆pENTR 1A-p16质粒的外源性合成的修饰后的p16 cDNA,取代目的载体pAd/CMV/V5-DEST中的ccdB基因,形成表达克隆pAd/CMV/V5-DEST-p16。测序证实质粒含有目的基因。PacI酶切后的重组腺病毒载体,通过脂质体2000介导,转染293A细胞,产生缺陷性的重组腺病毒。W estern blot分析显示在由重组腺病毒介导的野生型p16基因在肝癌细胞SMMC7721中能够表达蛋白。被感染的细胞的生长受抑制。结果构建了重组p16腺病毒载体,产生缺陷性的重组腺病毒,野生型p16基因对人肝癌细胞SMMC7721株有抑制作用。结论用通路克隆系统构建重组p16腺病毒载体稳定、可靠、方便,腺病毒能够介导野生型p16基因在肝癌细胞中表达,并抑制细胞的生长。

Aim To construct recombinant p16 Adenovirus vector and explore the suppressive function of wild type p16 gene in the growth of human heptocellular carcinoma cell line. Methods The sequence of the pl6-INK4 suppressor gene from human was artificially synthesized. And pENTR 1A p16 vectors, the entry clones, were generated by a serial tests. Along with the LR recombination reactions finished and the ccdB gene in the pAd/CMV/VS-DEST vector replaced by the p16-INK4, the pAd/CMV/VS-DEST-p16 vectors, the ex- pression clones, were created. The sequencing of the artificial synthesis sequence was right. The pAd/CMV/VS-DEST-p16 vectors digested by Pac I and purified were transfected into the 293A packaging cells by lipofection mediated transfection. Western blot indicated that P16 protein was expressed in the infected SMMC7721. Growth of the tumor cells infected by Ad/p16 virus was inhibited. Results Recombinant adenovirus AdV/p16 could infect the hepatocellular carcinoma SMMC7721 cells and have the suppressive function in the growth of tumor cells. Conclusions It is stable, reliable and convenient to construct the recombinant p16 Adenovirus vector with gateway cloning system. These results of suppressive function in the growth of tumor cells suggest that adenovirus is an efficient vector for mediating transfer and expression of the wild type gene p16 in human hepatocellular carcinoma cells.