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目的:构建同时携带低氧诱导因子-lα(HIF-lα)和角质细胞生长因子(KGF)的腺病毒载体(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cDNA作为模板,依据GeneBank公布的HIF-1αcDNA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pIRES2-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病毒骨架载体上构建携带HIF-1α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-lα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100 MOI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48h ELISA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:成功构建了腺病毒载体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。
OBJECTIVE: To construct adenovirus vector (pAdxsi-GFP-HIF-KGF) carrying both hypoxia inducible factor-1α (HIF-1α) and keratinocyte growth factor (KGF) and to observe its potential application in the prevention and treatment of lung injury. METHODS: The total RNA was extracted from A549 cells under hypoxia and reverse transcribed into cDNA. The primers were designed according to HIF-1αcDNA published by GeneBank and introduced into KpnI and BamHI sites respectively. The target gene HIF-1α Was ligated to the vector pShuttle-CMV-EGFP to construct the recombinant plasmid pShuttle-GFP-HIF. Then, the gene of KGF was amplified by PCR using the plasmid pIRES2-EGFP-KGF as a template and the primers of NheI and PmeI, and cloned into the recombinant plasmid pShuttle-GFP-HIF to obtain the shuttle plasmid pShuttle-GFP-HIF- KGF. Recombinant adenoviral vector pAdxsi-GFP-HIF-KGF carrying HIF-1α and KGF double genes was constructed by recombining the target sequence into pAdxsi virus backbone vector by bacterial recombination method. After detecting the titer of the recombinant adenovirus, A549 cells were transfected into human alveolar epithelial cells to detect the transfection expression of the target gene. Results: The recombinant adenovirus vector carrying pAdxsi-GFP-HIF-KGF carrying HIF-la and KGF genes was successfully constructed by sequencing and digestion of the constructed plasmid clones. The constructed recombinant adenovirus was of good purity and high titer. After transfection of A549 cells with pAdxsi-GFP-HIF-KGF at 100 MOI for 24 hours, the cells showed strong green fluorescence under fluorescence microscope, and the fluorescence was stronger at 48 hours. After 48 hours of transfection, The expression of HIF-1 protein was (56.36 ± 4.53) ng / mL and the expression of KGF protein was (60.20 ± 2.92) ng / mL. CONCLUSION: The adenoviral vector pAdxsi-GFP-HIF-KGF has been successfully constructed and its transfection efficiency and protein expression level of the target gene are high, which has the potential of further application in local application of lung injury. HIF-1 role in gene therapy drugs lay the foundation for the prevention and treatment of stress-induced acute lung injury in high altitude area to provide experimental basis.