双基因重组腺病毒表达载体pAdxsi—GFP-HIF—KGF的构建及表达 |
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引用本文: | 哈小琴,王鲲,张玉,邓芝云,董菊子,赵勇,彭俊华. 双基因重组腺病毒表达载体pAdxsi—GFP-HIF—KGF的构建及表达[J]. 西北国防医学杂志, 2013, 0(6): 501-503 |
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作者姓名: | 哈小琴 王鲲 张玉 邓芝云 董菊子 赵勇 彭俊华 |
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作者单位: | 兰州军区兰州总医院甘肃省干细胞与基因药物重点实验室,甘肃兰州730050 |
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基金项目: | 全军“十二五”面上基金资助项目(CWS11C229) |
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摘 要: | 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。
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关 键 词: | 低氧诱导因子-lα基因 角质细胞生长因子基因 腺病毒 构建 表达 |
Construction and expression of recombinant adenovirus pAdxsi - GFP - HIF - KGF containing hypoxia inducible factor gene and keratinocyte growth factor gene |
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Affiliation: | HA Xiao - qin, WANG Kun, ZHANG Yu, et al. ( Department of Clinical Laboratory, Key Laboratory of Stem Cell and Gene Medicine of Gansu Province, Lanzhou General Hospital of Lanzhou Military Area Command, PLA, Lanzhou 730050, China) |
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Abstract: | 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. |
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Keywords: | Hypoxia inducible factor let gene Keratinocyte growth factor gene Adenovirus Construc-tion Expression |
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