摘要
目的构建人缺氧诱导因子-1α(HIF-1α)基因的重组腺病毒载体,并观察其对HepG2细胞的转染情况。方法采用基因工程技术,经过亚克隆将人HIF-1α基因片段克隆至穿梭质粒pAdTrack-CMV上,利用pAdEasy系统进行细菌内同源重组后,经脂质体转染HEK293细胞,进行重组腺病毒的包装、扩增。采用PCR方法对重组腺病毒进行鉴定,利用穿梭质粒中带有绿色荧光蛋白GFP报告基因,进行病毒滴度的测定和对HepG2细胞感染效率的监测。结果酶切鉴定及PCR结果证明重组腺病毒载体成功,人HIF-1α重组腺病毒滴度达1.15×1010pfu/ml,阴性对照腺病毒的滴度为8×1010pfu/ml,并对HepG2细胞具有很强的感染力。结论应用细菌内同源重组法成功构建了含人HIF-1α基因的重组腺病毒载体。
Objective To construct the recombinant adenovirus of human HIF-1α gene and observe its ability to infect HepG2 hepatoma carcinoma cells. Methods The human HIF-1α gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria, and then was transfected into HEK293 cells with LipofectamineTM 2000. The target gene was detected by PCR. The titer and its infection rate were determined by using the green fluorescent protein（GFP） expression in the shuttle plasmid. Results Restriction endonuclease and PCR analyses confirmed that the HIF-1α gene was successfully inserted into the adenovirus vector. The titer of the human HIF-1α gene recombinant adenovirus was 1.15 × 10^10 pfu/ml, and that of the negative control recombinant adenovirus was 8 × 10^10 pfu/ml. The adenovirus had a strong effect on HepG2 hepatoma carcinoma cells. Conclusion The recombinant adenovirus containing human HIF-1α gene is successfully constructed by the method of homogenous recombinant in bacteria.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第20期2040-2043,共4页
Journal of Third Military Medical University
