本文關(guān)鍵詞：GAP-43腺相關(guān)病毒的制備及其在大鼠視網(wǎng)膜中的表達(dá)　出處：《福建醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： GAP-43 腺相關(guān)病毒 共轉(zhuǎn)染 AAV-293細(xì)胞 HT1080細(xì)胞

【摘要】： 目的:構(gòu)建攜帶生長相關(guān)蛋白43(GAP-43)基因的重組腺相關(guān)病毒,測(cè)定其感染滴度,并注射大鼠玻璃體腔,觀察其感染視網(wǎng)膜組織后的表達(dá)情況。探討通過腺相關(guān)病毒介導(dǎo)GAP-43基因治療視神經(jīng)及視網(wǎng)膜病變的可行性。 方法:克隆目的基因GAP-43片段,構(gòu)建重組質(zhì)粒pAAV-IRES-hrGFP-GAP-43。利用磷酸鈣法將pAAV-IRES-hrGFP-GAP-43、pAAV-RC和pHelper共轉(zhuǎn)染AAV-293細(xì)胞,構(gòu)建重組腺相關(guān)病毒( rAAV-GAP-43 ) ,經(jīng)“氯仿-PEG8000/NaCl-氯仿”的方法純化、濃縮,感染HT1080細(xì)胞,熒光顯微鏡下觀察熒光細(xì)胞數(shù),測(cè)定病毒滴度。大鼠玻璃體腔內(nèi)注射rAAV-GAP-43, 4W后眼球冰凍切片,熒光顯微鏡下觀察綠色熒光,western blot檢測(cè)視網(wǎng)膜組織中GAP-43蛋白的表達(dá)情況。 結(jié)果:成功構(gòu)建了pAAV-IRES-hrGFP-GAP-43重組質(zhì)粒;包裝制備了GAP-43腺相關(guān)病毒(rAAV-GAP-43),經(jīng)純化、濃縮后病毒滴度為3.0×109 /ml。rAAV-GAP-43注射大鼠玻璃體腔4W后,視網(wǎng)膜冰凍切片,熒光顯微鏡下可見到熒光表達(dá),western blot分析顯示視網(wǎng)膜組織高表達(dá)GAP-43蛋白。 結(jié)論:構(gòu)建的rAAV-GAP-43能感染視網(wǎng)膜組織,并在視網(wǎng)膜上成功表達(dá)GAP-43蛋白和綠色熒光蛋白。為GAP-43基因治療視神經(jīng)及視網(wǎng)膜病變奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R346

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本文編號(hào)：1356955