双基因重组腺病毒表达载体pAdxsi-GFP-HIF-KGF的构建及表达

Construction and expression of recombinant adenovirus pAdxsi-GFP-HIF-KGF containing hypoxia inducible factor gene and keratinocyte growth factor gene

目的:构建同时携带低氧诱导因子-1α(HIF-1α)和角质细胞生长因子(KGF)的腺病毒载体(pAdxsi-GFP-HIFKGF),观察其在A549中的表达。方法:从低氧处理A549细胞中PCR扩增HIF-1αcDNA,并连接到载体pShuttle-CMVEGFP上得到重组质粒pShuttle-GFP-HIF,同时将从质粒pIRES2-EGFP-KGF扩增的KGF基因也克隆其上,获得穿梭重组质粒pShuttle-GFP-HIF-KGF。再将其重组到pAdxsi病毒骨架载体上,构建携带HIF-1α和KGF双基因的重组腺病毒载体。观察重组腺病毒转染A549细胞后表达情况。结果:证实携带HIF-lα和KGF双基因的重组腺病毒载体pAdxsi-GFPHIF-KGF成功构建。其转染A549细胞后可有效表达HIF-1和KGF蛋白,48 h时HIF-1蛋白表达水平为(56.36±4.53)ng/ml,KGF蛋白表达水平为(60.20±2.92)ng/ml。结论:成功构建了双基因腺病毒载体pAdxsi-GFP-HIF-KGF,其可有效转染表达,具有进一步在肺损伤防治应用的前景。

Objective：To construct a recombinant adenovirus （pAdxsi - GFP - HIF - KGF） encoding human hypoxia inducible factor 1 ct gene （ HIF - 1 or） and human keratinocyte growth factor gene , and investigate its expres- sion in lung epithelium ceils. Methods： HIF - 1 oL gene from A549 in hypoxia condition and KGF gene from plas- mid pIRES2 - EGFP - KGF were subcloned into shuttle vector pShuttle - CMV - EGFP. A shuttle vector pShuttle - GFP - HIF - KGF was obtained. Then it was cotransformed into adenoviral backbone plasmid pAdxsi to make ho- mologous recombination with HIF - 1 ot gene and KGF gene. The recombinant adenovirus was infected with A549, and the expression level of HIF - 1 ct protein and KGF protein were evaluated by ELISA. Results：The recombinant adenovirus vector for HIF - 1 ct gene and KGF gene （ pAdxsi - GFP - HIF - KGF） was successfully constructed. The amount of HIF - 1 protein and KGF protein was （56.36 -＋ 4.53 ） ng/ml and （60.20 __ 2.92） ng/ml in superna- tant at 48 h after transfection, respectively. Conclusion： A recombinant adenovirus pAdxsi - GFP - HIF - KGF, encoding human hypoxia inducible factor 1 ot gene and keratinocyte growth factor gene, was constructed and ex- pressed successfully in A549 cells. It has further application prospect for the prevention and treatment of lung injury.