携带神经导向因子netrin-1基因的重组腺病毒构建  

Construction of recombinant adenovirus vector expressing the axonal attractant netrin-1

作　　者：姚丹[1] 李倩[2] 朱剑[2] 马建华[2] 毛晓明[2] 丁新生[2] 

机构地区：[1]南京医科大学第一附属医院神经内科,江苏南京210029 [2]南京医科大学附属南京第一医院内分泌科,江苏南京210006

出　　处：《南京医科大学学报（自然科学版）》2009年第3期356-360,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：南京市卫生局资助项目(ZKM06040)

摘　　要：目的:制备并构建神经导向因子netrin-1(NT-1)和EGFP双基因共表达的重组腺病毒载体,为后续的基因转染做准备。方法:采用基因克隆技术克隆目的基因NT-1,并将目的基因克隆入含有报告基因EGFP的穿梭载体pDC316质粒;构建的质粒pDC316-netrin经酶切及测序鉴定正确后,通过脂质体lipofectamine2000的介导与腺病毒包装质粒pBHGlox_E1.3Cre共转染至人胚肾细胞HEK293,经同源重组后得到携带鼠NT-1的重组腺病毒Ad5-netrin-CMV-EGFP。应用PCR鉴定重组腺病毒,空斑传代纯化病毒并反复冻融扩增病毒。以50%组织培养感染剂量法(TCID50)测定病毒滴度。结果:PCR鉴定证实重组腺病毒含有鼠NT-1基因,病毒滴度为5.2×109pfu/ml,EGFP活性检测腺病毒感染阳性细胞率在40%以上。结论:成功制备Ad5-netrin-CMV-EGFP腺病毒,为以后基因治疗研究打下基础。Objective：To construct and prepare the recombinant adenovirus vector co-expressing the axonal attractant netrin-1 and report gene EGFP as preparation for later use for genetic transfection. Methods：The netrin-1 was cloned by RT-PCR and the then subcloned into shuttle vector pDC316-CMV which carries the report gene EGFP. Subsequently, this newly constructed plasmid pDC316-netrin, after identification by nuclease digestion analysis and sequencing analysis, was transfected into human embryonic kidney cells HEK293 by lipofeetamine 2000 mediation, together with adenovirus-packaging plasmid pBHGlox_E1.3Cre. Based on homologous recombination of two plasmids within HEK293 cells, the recombinant adenovirus vector carrying netrin-1, AdS-netrin- CMV-EGFP, was created. Ad5-netrin-CMV-EGFP was subsequently identified by PCR, purified using repeated plaque passages, proliferated using freezing and melting with HEK293 cells, and titrated using 50% Tissue Culture Infective Dose （TCID50） assay. Results：The newly constructed recombinant adenovirus carrying rat netrin-1 was confirmed by PCR, and its titration value determined by TCIDS0 assay was 5.2 × 10^9pfu/ml. The positivity rate of adenovirus infected cells was over 40%. Conclusion：The recombinant adenovirus carrying rat netrin-1 was successfully constructed. The newly constructed can produce sufficiently high titration value with HEK293 cells, providing a reliable tool for genetic transfection in further gene therapy researches.

关 键 词：神经导向因子 腺病毒 基因重组 

分 类 号：Q784[生物学—分子生物学]