人p27^(kipl)可调控性重组腺病毒载体的构建及其在PC-3细胞中的表达

Construction of regulatory human p27~kipl recombinant adenovirus vector and its expression in human prostate cancer cell line PC-3

目的:体外构建人p27kipl可调控性重组腺病毒载体,并观察其在前列腺癌细胞系PC-3细胞中的表达及其对细胞生长的影响。方法:采用Ad-X Tet-Off表达系统,将人全长p27kipl cDNA经过穿梭载体,与可调控性腺病毒载体骨架连接,得到重组人p27kipl基因腺病毒(Ad-p27kipl),在HEK293细胞包装、扩增,获得高滴度病毒,采用空斑试验测定病毒滴度,体外转染PC-3细胞,RT-PCR、Western杂交分别检测目的基因不同水平的表达,并通过MTT法观察转染前后细胞增殖力的变化。Ad-X-TRE-β gal病毒作为对照。结果:构建的Ad-p27kipl病毒滴度为1.2×109 pfu/ml,RT-PCR、Western杂交检测有特异的高表达,转染后对细胞的生长有抑制作用。结论:体外连接法成功地构建携带人p27kipl基因的可调控性重组腺病毒载体,在人前列腺癌细胞系PC-3中有高表达并对细胞生长有抑制作用。

Objective:To construct regulatory human p27kipl recombinant adenovirus vector and analyze its expression in human prostate cancer cell line PC-3 and its effects on cell proliferation. Methods: The full-length cDNA encoding p27*'v] gene was inserted into pTRE-Shuttle vector. Then the expression cassette containing tetracycline-responsive element, which regulating the expression of inserted genes, was excised with restriction enzymes PI-Sce I and I-Ceu I and ligated to the backbone of the Adeno-X viral DNA. The recombinant adenovirus plasmid was identified by restriction enzymes excising analysis and PCR. The replication-incompetent recombinant adenovirus was packaged and propagated in HEK293 cells. The viral titer was examined by plaque assay; the mRNA and protein expressions of p27kipl were determined by RT-PCR and Western bolt respectively. MTT assay was used to assess the effect of p27kipl on cell proliferation. Ad-X-TRE (3 gal virus was used as control. Results: The viral liter of Ad-p27kipl was 1. 2X 109 pfu/ml and the P27 protein expression was positive and high. The growth of PC-3 cells treated with Ad-p27kipl was significanlly inhibiled compared with that treated with control adenovirus. Conclusion; The human p27kipl recombinanl adenovirus has been successfully built and is highly expressed in human prostate cancer cell line PC-3. Adenoviral-mediated expression of human p27kipl can inhibit the proliferation of human proslate cancer cell lines PC-3.