人肝细胞生长因子基因重组腺病毒载体的构建  

Construction of adenovirus- recombined vector of hepatocyte growth factor

作　　者：王祥[1] 刘本德[1] 曾秋棠[1] 阮秋蓉[2] 

机构地区：[1]华中科技大学协和医院心内科华中科技大学同济医学院心血管病研究所,武汉430022 [2]华中科技大学同济医学院病理学系

出　　处：《临床心血管病杂志》2006年第4期238-241,共4页Journal of Clinical Cardiology

基　　金：湖北省自然科学基金(No:2004ABA222)

摘　　要：目的:利用ADEASYTM系统,构建高效而又安全的肝细胞生长因子(HGF)基因腺病毒载体(AdHGF),以研究HGF基因在心脏内的表达。方法:从人胎肝中抽提总RNA,并以该肝细胞总RNA为模版,利用合成的1对引物,采用RTPCR技术获得HGF基因的cDNA,并引入酶切位点,先转入感受态质粒,再转化大肠杆菌进行扩增,筛选阳性菌落,经酶切及测序,证实目的HGF基因已经转入,再将该基因转入pUC18质粒,pUC18质粒与AdEASY质粒进行同源重组,用PacⅠ酶切成线性化DNA,利用脂质胺转染293细胞,经3轮扩増,制备高效表达并用绿色荧光标志AdHGF。并将该载体作实验组,与HGF组作比较,观察对VEC的抗凋亡作用。结果:在荧光显微镜发现293细胞发光率为100%,AdHGF的HGFcDNA经过测序鉴定与基因库序列一致。结论:AdHGF的成功构建,为冠心病转基因治疗的基础与临床研究奠定了基础。Objective：To construct high efficient and safe adenovirus vector of hepatocyte growth factor （Ad-HGF）gene recombination with AD-EASYTM system in order to research the expression of HGF gene in the heart. Method..To extract total RNA from human fetal liver, take the total RNA of hepatocyte as model, use a pair of synthetic primers,adopt RT-PCR technique to obtain cDNA of HGF gene and insert enzyme cutting site, turn to competence plasmid first, and then transfer the colibacillus to amplify, screen the positive colony,ensure the targeted HGF gene being transferred after enzyme cutting and sequencing, then transfer the gene into pUC18 plasmid, carry out homologous recombination with pUC18 plasmid and AD-EASY plasmid, enzyme cutting into linearization DNA with PacI, use fatty amine to transfect 293 cell, amplify three times, Ad-HGF gene recombination labelled by fluorescence was prepared with high effective expression. Result： HGF full length cDNA has been cloned and recombinant HGF adenovirus vector was constructed successfully, as shown by PCR, restricting endonuclease digestion analysis and fluorescence detection. It was found that 293 cell luminance was 100 % under the fluorescence microscope. Conelusion：A recombinant adenovirus vector of HGF was constructed successfully which may be a potential basis for the gene therapy of coronary artery disease and established a foundation for further study.

关 键 词：肝细胞生长因子 腺病毒 载体 基因治疗 

分 类 号：R373.1[医药卫生—病原生物学]