双基因重组腺病毒表达载体pAdxsi—GFP-HIF—KGF的构建及表达

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—HIF—KGF），观察其在A549中的表达。方法：从低氧处理A549细胞中PCR扩增HIF—1αDNA，并连接到载体pShuttle—CMV—EGFP上得到重组质粒pShuttle—GFP—HIF，同时将从质粒pIRES2一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-1仅和KGF双基因的重组腺病毒载体。观察重组腺病毒转染3_549细胞后表达情况。结果：证实携带HIF—1α和KGF双基因的重组腺病毒载体pAdxsi—GFP—HIF—KGF成功构建。其转染A549细胞后可有效表达HIF一1和KGF蛋白，48h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。

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

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.