文章摘要
姚 丹,李 倩,朱 剑,马建华,毛晓明,丁新生.携带神经导向因子netrin-1基因的重组腺病毒构建[J].南京医科大学学报,2009,29(3):356~360
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
投稿时间:2008-08-13  
DOI:10.7655
中文关键词: 神经导向因子  腺病毒  基因重组
英文关键词: the neural guidance factor  adenovirus vector  gene recombinant
基金项目:南京市卫生局资助项目(ZKM06040)
作者单位
姚 丹 南京医科大学第一附属医院神经内科,江苏 南京 210029 
李 倩 南京医科大学附属南京第一医院内分泌科,江苏 南京 210006 
朱 剑 南京医科大学附属南京第一医院内分泌科,江苏 南京 210006 
马建华 南京医科大学附属南京第一医院内分泌科,江苏 南京 210006 
毛晓明 南京医科大学附属南京第一医院内分泌科,江苏 南京 210006 
丁新生 南京医科大学第一附属医院神经内科,江苏 南京 210029 
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中文摘要:
      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?
英文摘要:
      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 lipofectamine 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, Ad5-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 TCID50 assay was 5.2 × 109pfu/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.
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