| 人胆固醇酯水解酶重组腺病毒载体的构建 |
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| 引用本文： | 张兴海,罗俊生,霍晓川,顾立学,关宁,冯旭.人胆固醇酯水解酶重组腺病毒载体的构建[J].山东医药,2011,51(10):17-18. |
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| 作者姓名： | 张兴海 罗俊生 霍晓川 顾立学 关宁 冯旭 |
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| 作者单位： | 辽宁医学院,辽宁锦州,121001 |
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| 基金项目： | 国家自然科学基金资助项目，辽宁省自然科学基金项目，辽宁省教育厅高等学校科研项目计划 |
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| 摘 要： | 目的构建人胆固醇酯水解酶（hCEH）基因重组腺病毒载体,并在人胚肾细胞（HEK293）中扩增。方法将hCEH基因整合入腺病毒穿梭质粒pAdTrack-CMV中,构建重组穿梭质粒pAdTrack-CMV-hCEH,线性化后与pAdeasy-1在大肠杆菌BJ5183中进行同源重组,获得含目的基因的重组质粒pAd-hCEH,酶切后用脂质体转染HEK293,包装成重组体腺病毒Ad-hCEH,经扩增和纯化得到高滴度重组病毒。采用PCR法进行鉴定,绿色荧光蛋白（GFP）追踪,荧光显微镜下观察并计算Ad-hCEH病毒滴度。结果重组穿梭质粒及其载体经鉴定后,电泳结果与预期相符。Ad-hCEH-EGFP感染HEK293后,随时间增加,HEK293表达绿色荧光的量也增加。hCEH碱基序列经测序与Genebank中已知序列完全吻合。Ad-hCEH电泳结果显示1 700 bp附近有一条带,与目的片段相符合。最终测得Ad-hCEH滴度为1.2×1010 pfu/ml。结论成功构建了携带hCEH基因的重组腺病毒载体。
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| 关 键 词： | 胆固醇酯水解酶 腺病毒 质粒 人胚肾细胞 腺病毒载体系统 |
Construction of human CEH recombinant adenovirus vector |
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| ZHANG Xing-hai,LUO Jun-sheng,HUO Xiao-chuan,GU Li-xue,GUAN Ning,FENG Xu.Construction of human CEH recombinant adenovirus vector[J].Shandong Medical Journal,2011,51(10):17-18. |
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| Authors: | ZHANG Xing-hai LUO Jun-sheng HUO Xiao-chuan GU Li-xue GUAN Ning FENG Xu |
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| Institution: | ZHANG Xing-hai,LUO Jun-sheng,HUO Xiao-chuan,GU Li-xue,GUAN Ning,FENG Xu(Liaoning Medical University,Jinzhou 121001,P.R.China) |
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| Abstract: | Objective To construct hCEH recombinant replication deficient adenovirus vector pAd-hCEH amplify in HEK293 cells.Methods hCEH gene was cloned into shuttle vector pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-hCEH,and the resultant plasmid was linearized and was subsequently homogenous recombination with pAdeasy-1 in E.coli BJ5183 cells.Recombinants were screened and were identified by observation of green fluorescent protein(GFP) and PCR.The recombinant plasmid was transfected into HEK293 to p... |
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| Keywords: | cholesterol ester by drolase adenovirus plasmid human embryonic kidney cell adenovirus vector system |
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