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人表皮生长因子慢病毒载体的构建及表达 被引量：3

Construction and expression of lentivirus vector for human epidermal growth factor
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摘要 目的:构建人表皮生长因子(hEGF)慢病毒(LV)载体。方法:酶切并收集pcDNA3.1-hEGF载体中的hEGF目的基因片段,克隆进入含有绿色荧光蛋白(GFP)基因的慢病毒载体内,将所得重组体LV-GFP-hEGF与辅助载体pΔ8.2和pVSVG共同转染人胚肾293T细胞,进行病毒包装。收集上清液,按按10-1～10-7倍比稀释后培养,检测病毒滴度;裂解细胞,进行Western Blot分析。结果:PCR扩增、HindⅢ酶切鉴定及测序结果均证实LV-GFP-hEGF构建成功。将LV-GFP-hEGF转染293T细胞,紫外光下检测可见绿色荧光;LV滴度达5×108TU/mL,转染效率>75%,Western Blot证实转染细胞可表达hEGF。结论:成功构建了含有GFP报告基因的hEGF慢病毒载体,包装后的慢病毒可在293T细胞内高效表达。 Aim：To construct lentivirus vector（LV） of human epidermal growth factor（hEGF）.Methods：hEGF target sequence in pcDNA-hEGF 3.1 vector was cut,extracted and inserted into LV which contained green fluorescence protein（GFP） gene,then the recombinant LV-GFP-hEGF was cotransfected into human embryonic kidney 293T cells along with assisting vectors pΔ8.2 and pVSVG to package the virus.The supernatant was extracted,attenuated according to different titer,and cultivated,the virus titer was detected,and the cells were clearaged and analyzed with Western Blot.Results：LVGFP-hEGF was constructed successfully,and could express in 293T cells.The virus titer was 5 × 108 TU/mL.Conclusion：LV which contained GFP and hEGF gene has been constructed successfully,and the packaged lentivirus could express effectively in 293T cells.
作者 张丽梅 冯龙 张艳敏 马晶 赵国强 安玉会 鲍玉洲
机构地区 河南省眼科研究所分子生物室 郑州大学基础医学院生物化学与分子生物学教研室 郑州大学基础医学院微生物学与免疫学教研室
出处 《郑州大学学报（医学版）》 CAS 北大核心 2010年第4期603-606,共4页 Journal of Zhengzhou University(Medical Sciences)
关键词 人表皮生长因子 慢病毒载体 基因构建 human epidermal growth factor lentivirus vector gene construct
分类号 Q789 [生物学—分子生物学]
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参考文献9

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共引文献2

同被引文献34

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二级引证文献7

郑州大学学报（医学版）
2010年 第4期

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