嗜肝细胞重组腺病毒基因治疗载体的构建

发布时间：2018-05-27 17:31

本文选题：腺病毒 + 乙型肝炎病毒　；参考：《重庆医科大学》2010年硕士论文

【摘要】： 目的:重组腺病毒(rAd)是常用的基因治疗载体,但它缺乏嗜肝性,本课题通过改造rAd纤毛结构,拟构建出具有嗜肝性的rAd载体,用于肝病靶向基因治疗。方法:以目前常用的rAd高表达载体系统AdEasy System为基础,用乙型肝炎病毒(HBV)衣壳蛋白中具有天然嗜肝性的肽段PreS1的21-47位氨基酸的编码基因(preS1),定点克隆至AdEasy System中骨架质粒pAdEasy-1的HI Loop编码基因位点,构建出重组骨架质粒pAdEasy-1-preS1 ,再用pAdEasy-1-preS1与穿梭质粒pShuttle- IRES-hrGFP-1同源重组成病毒质粒pAd-pres1,经转染HEK293细胞后包装生成重组腺病毒基因治疗载体rAd-PreS1。1 结果:克隆了preS1基因片段的腺病毒重组子经测序显示基因序列正确;病毒质粒转染HEK293细胞,一周后进行荧光观察发现其表达出荧光蛋白,提示有腺病毒生成;以重组腺病毒基因为模板行PCR检测能提取嗜肝性基因片段preS1;抽提重组腺病毒蛋白用免疫印迹法能检测到目标蛋白PreS1(21-47)的表达,提示有重组腺病毒基因治疗载体rAd-PreS1的生成;用构建的rAd-PreS1感染多种细胞,病毒滴度检测显示对肝细胞具有相对嗜向性。结论:在rAd纤毛球域HI Loop的编码基因位点插入HBV表面蛋白中嗜肝性片段PreS1的21-47位氨基酸的编码基因preS1,经HEK293细胞包装生成的重组腺病毒基因治疗载体rAd-PreS1,通过体外细胞实验显示对肝细胞具有相对嗜向性。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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