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| PCR靶向克隆法构建携带人类神经生长因子β基因的含有EGFP的重组腺病毒穿梭质粒 | |
| 引用本文： | 谢华,王清秀,王家宁,肖敏,黄永章.PCR靶向克隆法构建携带人类神经生长因子β基因的含有EGFP的重组腺病毒穿梭质粒[J].武汉大学学报(医学版),2009,30(2). |
| 作者姓名： | 谢华 王清秀 王家宁 肖敏 黄永章 |
| 作者单位： | 1. 郧阳医学院附属太和医院急诊科湖北,十堰,442000 2. 同济大学附属东方医院麻醉科,上海,200120 3. 郧阳医学院附属人民医院临床医学研究所,湖北,十堰,442000 |
| 基金项目： | 湖北省教育厅重大项目(编号:Z200724001);;同济大学附属东方医院科研项目(编号:DF2007R02);;上海市浦东新区优秀学科带头人培养计划(编号:PWRd2007-04) |
| 摘 要： | 目的:在靶向克隆酶的作用下,利用PCR靶向克隆构建携带人神经生长因子的重组腺病毒穿梭质粒pShuttle-hNGFβ-EGFP。方法:按PCR引物设计原则设计两条引物,分别在上下游引物的5′端加上12个碱基与线性化载体切口两端同源载体序列,引物合成后PCR反应扩增目的基因hNGF-β,提纯后与EcoRⅤ酶切的穿梭质粒pShuttle-IRES-hrEGFP在靶向克隆酶(LPReccoenzyme)的作用下37℃孵育15min,转化感受态大肠杆菌,碱裂解法制备及纯化质粒DNA。结果:EcoRⅤ酶切鉴定证实pShuttle-hNGFβ-EGFP构建成功。结论:在不需要限制性内切酶的情况下用靶向克隆酶连接可快速、准确得到穿梭质粒pShuttle-hNGFβ-EGFP。 |
| 关 键 词： | 靶向克隆 穿梭质粒 神经生长因子 |
Construction of Recombinant Human Beta-Nerve Growth Factor Adenoviral Shuttle Plasmid with Enchanced Green Fluorescent Protein by PCR Target Clone |
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| XIE Hua,WANG Qingxiu,WANG Jianing,XIAO Ming,HUANG Yongzhang.Construction of Recombinant Human Beta-Nerve Growth Factor Adenoviral Shuttle Plasmid with Enchanced Green Fluorescent Protein by PCR Target Clone[J].Medical Journal of Wuhan University,2009,30(2). | |
| Authors: | XIE Hua WANG Qingxiu WANG Jianing XIAO Ming HUANG Yongzhang |
| Institution: | XIE Hua1,WANG Qingxiu2,WANG Jianing3,XIAO Ming1,HUANG Yongzhang31Dept.of Emergency,Taihe Hospital,Yunyang Medical College,Shiyan 442000,China2Dept.of Anesthesiology,Dongfang Hospital,Tongji University,Shanghai 200120,China3Institute of Clinical Medicine,People's Hospital,China |
| Abstract: | Objective: To construct pShuttle-hNGFβ-EGFP by PCR target clone with target clone enzyme.Methods: According to the principle of designing PCR primer,two primers were designed and a sequence of twelve bases of the shuttle vector was added at the 5'-end respectively.After the two primers were synthesized,hNGFβ was cloned by PCR and shuttle vector was digested with EcoRⅤ,mixed with hNGFβ,shuttle vector and target clone enzyme(LP Recco enzyme),and incubated at 37℃ for 15 minutes,and then was transfected into E.coli immediately.Alkaline lysis method was adopted to prepare and purifiy the plasmid DNA.Results: The construction of pShuttle-hNGFβ-hrEGFP-2 was identified by the digestion with EcoRⅤ.Conclusion: Gene clone with target clone enzyme can connect purpose gene with shuttle vector quickly and exactly without restriction enzyme. |
| Keywords: | Target Clone Shuttle Vector Nerve Growth Factor |
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