GAP-43腺相关病毒的制备及其在大鼠视网膜中的表达

发布时间：2017-12-30 23:12

本文关键词：GAP-43腺相关病毒的制备及其在大鼠视网膜中的表达　出处：《福建医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:构建携带生长相关蛋白43(GAP-43)基因的重组腺相关病毒,测定其感染滴度,并注射大鼠玻璃体腔,观察其感染视网膜组织后的表达情况。探讨通过腺相关病毒介导GAP-43基因治疗视神经及视网膜病变的可行性。方法:克隆目的基因GAP-43片段,构建重组质粒pAAV-IRES-hrGFP-GAP-43。利用磷酸钙法将pAAV-IRES-hrGFP-GAP-43、pAAV-RC和pHelper共转染AAV-293细胞,构建重组腺相关病毒( rAAV-GAP-43 ) ,经“氯仿-PEG8000/NaCl-氯仿”的方法纯化、浓缩,感染HT1080细胞,荧光显微镜下观察荧光细胞数,测定病毒滴度。大鼠玻璃体腔内注射rAAV-GAP-43, 4W后眼球冰冻切片,荧光显微镜下观察绿色荧光,western blot检测视网膜组织中GAP-43蛋白的表达情况。结果:成功构建了pAAV-IRES-hrGFP-GAP-43重组质粒;包装制备了GAP-43腺相关病毒(rAAV-GAP-43),经纯化、浓缩后病毒滴度为3.0×109 /ml。rAAV-GAP-43注射大鼠玻璃体腔4W后,视网膜冰冻切片,荧光显微镜下可见到荧光表达,western blot分析显示视网膜组织高表达GAP-43蛋白。结论:构建的rAAV-GAP-43能感染视网膜组织,并在视网膜上成功表达GAP-43蛋白和绿色荧光蛋白。为GAP-43基因治疗视神经及视网膜病变奠定了基础。
[Abstract]:Objective: to construct a recombinant adeno-associated virus carrying growth associated protein 43 (GAP-43) gene and to determine its infection titer and inject it into rat vitreous cavity. To investigate the expression of adeno-associated virus mediated GAP-43 gene in the treatment of optic nerve and retinopathy. Methods: the GAP-43 fragment of the target gene was cloned. The recombinant plasmid pAAV-IRES-hrGFP-GAP-43was constructed. PAAV-IRES-hrGFP-GAP-43 was obtained by calcium phosphate method. PAAV-RC and pHelper co-transfected AAV-293 cells to construct recombinant adeno-associated virus (rAAV-GAP-43). The HT1080 cells were purified by chloroform-PEG8000 / NaCl-chloroform, concentrated, infected with HT1080 cells, and the number of fluorescent cells was observed under fluorescence microscope. After intravitreal injection of rAAV-GAP-43,4W, the green fluorescence was observed under fluorescence microscope. Western blot was used to detect the expression of GAP-43 protein in retina. Results: pAAV-IRES-hrGFP-GAP-43 recombinant plasmid was successfully constructed. GAP-43 adeno-associated virus rAAV-GAP-43 was prepared and purified. The titer of concentrated virus was 3.0 脳 109 / ml. RAAV-GAP-43 was injected into the vitreous cavity of rats for 4 W. Western blot analysis showed high expression of GAP-43 protein in retina. Conclusion: the constructed rAAV-GAP-43 can infect retinal tissue. GAP-43 protein and green fluorescent protein were successfully expressed in the retina, which laid a foundation for the treatment of optic nerve and retinopathy by GAP-43 gene.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

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