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  

Construction of Recombinant Human Beta-Nerve Growth Factor Adenoviral Shuttle Plasmid with Enchanced Green Fluorescent Protein by PCR Target Clone

作　　者：谢华[1] 王清秀[2] 王家宁[3] 肖敏[1] 黄永章[3] 

机构地区：[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

出　　处：《武汉大学学报（医学版）》2009年第2期212-215,I0002,共5页Medical Journal of Wuhan University

基　　金：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。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.

关 键 词：靶向克隆 穿梭质粒 神经生长因子 

分 类 号：R378.21[医药卫生—病原生物学] R734.2[医药卫生—基础医学]