| 【摘要】 目的:克隆出大鼠生长相关蛋白（rGAP-43）的基因片段,并以腺相关病毒（AAV）为载体构建出rGAP-43的表达系统，为应用病毒介导基因转移的方法来研究GAP-43对大鼠视网膜中各类细胞的作用及为以后进行基因治疗的研究奠定基础。方法: 1.经RT-PCR法,获得新生SD大鼠脑组织的cDNA文库，设计引物，从中扩增出rGAP-43基因的ORF（开放读码框）区片段,克隆入T载体,经序列测定正确后，再大量扩增rGAP-43基因片段。2.将序列测定无误后的目的基因—rGAP-43经BamHⅠ/SalⅠ酶切，连接上经同样酶切的含GFP（绿色荧光蛋白）报告基因的AAV质粒载体（AAV-IRES-GFP）。随后针对重组表达质粒AAV-GFP-rGAP-43进行酶切、序列测定等结构上的鉴定。 3.将重组子及AAV转染需要的辅助质粒pHelper、pAAV-RC经脂质体共转染培养好的AAV-293细胞，进行荧光表达的检测以及病毒原液的收集，病毒感染性滴度的测定等。结果: 1.克隆出的大鼠GAP-43基因的ORF区片段以及构建的AAV-GFP-GAP-43重组子，两者经酶切鉴定与序列测定均证实结构正确。2.重组表达质粒转染后的AAV-293细胞可检测到标记基因绿色荧光的表达，根据表达荧光的细胞数得到重组病毒的感染性滴度约为106 particles/ml。结论: 成功克隆了大鼠GAP-43基因并构建了以腺相关病毒为载体、可产生绿色荧光的rGAP-43表达质粒，可以应用表达质粒来更好地研究rGAP-43在视网膜、视神经的各种阶段中所起的作用。
【关键词】 生长相关蛋白; 分子克隆; 腺相关病毒载体
Construction of recombinant adeno-associated virus vector expressing the rat GAP-43 gene
ZHU yihua， LIN shibin， YANG juhua ， LIN fasen Ophthalmic Center ,The First Affiliate Hospital of Fujian Medical University, Fuzhou 350005, China
Corresponding author: LIN shibin, E-mail:bob239@sina.com
【Abstract】 Objective To construct the recombinant adeno-associated virus (rAAV) vector expressing the rat GAP-43 gene. Methods 1.The rGAP-43 ORF (open reading frame) cDNA was generated by RT-PCR using specific primers. This fragment was cloned into the pUCm-T vector and the result of sequence analysis was exact. Then the gene fragment was amplified by PCR through a pair of primers designed. 2. Then the rGAP-43 ORF gene fragment was digested with BamHⅠ/SalⅠ and cloned into plasmid AAV-IRES-GFP digested with the same enzymes, to construct the recombinant AAV-GFP-GAP-43 plasmid containing the reporter gene GFP (enhanced green fluorescence protein) and that was verified by DNA sequencing and enzyme digestion. 3. The recombinant expression plasmid was co-transfect into the AAV-293 cells with pAAV -RC and pHelper by lipofectamine transfection method, then to observe the expression of GFP under a fluorescence microscope and to package rAAV vector and to measure the infectivity titre of virus. Results 1.Both rGAP-43 ORF gene fragment and the recombinant AAV-GFP-GAP-43 plasmid were verified by enzyme digestion and DNA sequencing for correct reading frame and orientation. 2. GFP expression in co-transfected AAV-293 cells was observed and the titer of rAAV is 106particles/ml measured by that cells expressing green fluorescence were counted and compared with the total number of cells. Conclusion Successful construction of rAAV vector expressing the rat GAP-43 gene and GFP gene will provide a foundation for further research of rGAP-43 function on the mouse's vision system by the method of viral mediated gene transfer.
【Key words】 Growth associated protein-43；molecular cloning；adeno-associated virus vector
|
