本文選題：腺病毒 + 乙型肝炎病毒��； 參考：《重慶醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的:重組腺病毒(rAd)是常用的基因治療載體,但它缺乏嗜肝性,本課題通過改造rAd纖毛結(jié)構(gòu),擬構(gòu)建出具有嗜肝性的rAd載體,用于肝病靶向基因治療。 方法:以目前常用的rAd高表達(dá)載體系統(tǒng)AdEasy System為基礎(chǔ),用乙型肝炎病毒(HBV)衣殼蛋白中具有天然嗜肝性的肽段PreS1的21-47位氨基酸的編碼基因(preS1),定點(diǎn)克隆至AdEasy System中骨架質(zhì)粒pAdEasy-1的HI Loop編碼基因位點(diǎn),構(gòu)建出重組骨架質(zhì)粒pAdEasy-1-preS1 ,再用pAdEasy-1-preS1與穿梭質(zhì)粒pShuttle- IRES-hrGFP-1同源重組成病毒質(zhì)粒pAd-pres1,經(jīng)轉(zhuǎn)染HEK293細(xì)胞后包裝生成重組腺病毒基因治療載體rAd-PreS1。1 結(jié)果:克隆了preS1基因片段的腺病毒重組子經(jīng)測序顯示基因序列正確;病毒質(zhì)粒轉(zhuǎn)染HEK293細(xì)胞,一周后進(jìn)行熒光觀察發(fā)現(xiàn)其表達(dá)出熒光蛋白,提示有腺病毒生成;以重組腺病毒基因?yàn)槟０逍蠵CR檢測能提取嗜肝性基因片段preS1;抽提重組腺病毒蛋白用免疫印跡法能檢測到目標(biāo)蛋白PreS1(21-47)的表達(dá),提示有重組腺病毒基因治療載體rAd-PreS1的生成;用構(gòu)建的rAd-PreS1感染多種細(xì)胞,病毒滴度檢測顯示對(duì)肝細(xì)胞具有相對(duì)嗜向性。 結(jié)論:在rAd纖毛球域HI Loop的編碼基因位點(diǎn)插入HBV表面蛋白中嗜肝性片段PreS1的21-47位氨基酸的編碼基因preS1,經(jīng)HEK293細(xì)胞包裝生成的重組腺病毒基因治療載體rAd-PreS1,通過體外細(xì)胞實(shí)驗(yàn)顯示對(duì)肝細(xì)胞具有相對(duì)嗜向性。
[Abstract]:Objective: recombinant adenovirus rAdis is a commonly used gene therapy vector, but it lacks lipophilia. By modifying the structure of rAd cilium, we intend to construct a hepatophilic rAd vector for targeted gene therapy of liver diseases. Methods: based on AdEasy System, a high expression vector system of rAd, which is commonly used at present, The 21-47 amino acid encoding gene of PreS1, a naturally hepatophilic peptide in hepatitis B virus (HBV) capsid protein, was cloned into the HI Loop coding site of the skeleton plasmid pAdEasy-1 in AdEasy System. The recombinant skeleton plasmid pAdEasy-1-preS1 was constructed. The recombinant adenovirus gene therapy vector (rAd-PreS1.1) was formed by the homologous recombination of pAdEasy-1-preS1 and shuttle plasmid pShuttle- IRES-hrGFP-1 into pAd-pres1. After transfection into HEK293 cells, the recombinant adenovirus gene therapy vector rAd-PreS1.1 was formed. Results: the recombinant adenovirus fragment cloned from preS1 gene was sequenced correctly, and the recombinant plasmid was transfected into HEK293 cells. After a week of fluorescence observation, it was found that the recombinant adenovirus expressed fluorescent protein, indicating adenovirus formation. The recombinant adenovirus gene was used as template for PCR detection to extract the hepatophilic gene fragment preS1, the recombinant adenovirus protein was extracted by Western blotting to detect the expression of the target protein PreS1m21-47), indicating the production of recombinant adenovirus gene therapy vector rAd-PreS1. RAd-PreS1 was used to infect many kinds of cells, and the virus titer test showed that it had relative tropism to hepatocytes. Conclusion: the coding gene site of HI Loop in rAd ciliated sphere domain is inserted into the encoding gene preS1 of 21-47 amino acid of PreS1 in HBV surface protein. The recombinant adenovirus gene therapy vector rAd-PreS1 is produced by HEK293 cell packaging. The recombinant adenovirus gene therapy vector rAd-PreS1 was obtained by in vitro refinement. Cell experiments showed that the liver cells had a relative tropism.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346

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本文編號(hào)：1943147