低氧诱导因子和角质细胞生长因子双基因重组腺病毒的构建及其在肺泡上皮细胞中的表达

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）N腺病毒载体（pAdxsi-GFP-HIF-KGF），观察其在防治肺损伤潜在的应用前景。方法：低氧处理A549细胞后提取总RNA并逆转录为eDNA作为模板，依据GeneBank公布的HIF-1α cDNA设计引物，并分别引入KpnI和BamHI酶切位点，PCR扩增后将目的基因HIF-1α连接到载体pShuttle-CMV-EGFP上，构建重组质粒pShuttle-GFP—HIF。然后以质粒plRES2-EGFP-KGF为模板，用引入NheI和PmeI酶切位点的引物PCR扩增KGF基因并克隆到重组质粒pShuttle-GFP-HIF上，获得穿梭质粒重组质粒pShuttle—GFP-HIF—KGF。采用细菌内重组方法将目的序列重组到pAdxsi病毒骨架栽体上构建携带HIF．10t和KGF双基因的重组腺病毒载体pAdxsi-GFP-HIF-KGF。检测重组腺病毒滴度后，转染人肺泡上皮细胞A549，检测目的基因的转染表达。结果：通过对构建质粒克隆进行测序及酶切，证实携带HIF—lot和KGF双基因的重组腺病毒载体pAdxsi-GFP-HIF-KGF构建成功，且构建的重组腺病毒纯度好、滴度高。用pAdxsi-GFP-HIF-KGF以100MOI转染A549细胞后24h后在荧光显微镜下可观察到细胞有较强的绿色荧光表达，48h时荧光更强；转染48hELISA法检测培养上清中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：成功构建了腺病毒栽体pAdxsi-GFP-HIF-KGF，其转染效率及目的基因的蛋白表达水平较高，具有潜在的进一步在肺损伤局部应用的前景，为后期制备可以同时发挥KGF、HIF-1作用的基因治疗药物打下基础，同时为高海拔地区应激性急性肺损伤的有效防治提供实验基础。

Construction of Recombinant Adenovirus pAdxsi-GFP-HIFContaining Hypoxia Inducible Factor Gene and its Expression in Endothelial Cells

Objective： To construct a recombinant adenovirus （pAdxsi-GFP-HIF） encoding human hypoxia inducible factor lαgene （HIF-lα） and human keratinocyte growth factor gene, and investigate its expression in lung epithelium cells. Methods： H1F-lagene was obtained from human lung cancer cell line A549 which was cultured in hypoxia condition by RT-PCR. The HIF-letgene was subcloned into shuttle vector pShuttle-CMV-EGFP at KpnI and BamI-II sites. And the KGF gene from plasmid plRES2-EGFP-KGF by PCR was al- so subcloned into the shuttle vector at NheI and PmeI sites. A shuttle vector pShuttle-GFP-HIF-KGF was obtained. After identified with restriction enzymes, plasmid pShuttle-GFP-HIF-KGF was linearized by digestion with restriction endonuclease I-CeuI and I-SceI, and was subsequently cotransformed into E.coli DH5a with adenoviral backbone plasmid pAdxsi to make homologous recombination. After linearized by PacI, the homologous recombinant adenovirus plasmid was transfected into 293 cells to package and amplify. The recombi- nant adenovirus was infected A549, and the expression level of HIF-hxprotein and KGF protein were evaluated by ELISA. Results： The recombinant adenovirus vector for HIF-lαxgene and KGF gene （pAdxsi-GFP-HIF-KGF）was successfully constructed and amplified with titer of 2.59× 10^10 pfu/mL. The green fluorescence protein could be observed under fluorescent microscope in A549 at 24h after transfec- tion and with a stronger degree after 48h. The amount of HIF-1 protein and KGF protein was 56.36± 4.53 ng/mL and 60.20± 2.92 ng/ mL in supernatant at 48h after transfection,respectively. Conclusion： A recombinant adenovirus pAdxsi-GFP-HIF-KGF, encoding human hypoxia inducible factor lαgene and keratinocyte growth factor gene, was constructed in vitro and expressed successfully in A549 cells. It provides the material basis for further studying the biologic function and potential application of the recombinant adenovirus, in order to prepare for gene drug can play a role of KGF and HIF-1 and provide a experimental basis for effective prevention of acute lung injury at high altitudes.