乙肝病毒核心蛋白基因工程改造及其生物学功能鉴定

发布时间：2018-03-18 21:22

本文选题：乙型肝炎病毒　切入点：核心蛋白　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的探讨基于乙肝病毒核心蛋白（HBc）的改造位点及形式，使形成携带细胞穿膜肽或配体的核心颗粒，以作为HBV感染模型改造的基础。方法 1用不依赖酶切连接的分子克隆方法（RLIC）构建HBc、HBV1.1c-及各种HBc改造质粒。 2将各种HBc重组体与HBV1.1c-共转染HEK293细胞，通过提取细胞内核心颗粒DNA，Southern blot检测DNA中间体，来判断相应基因工程改造HBc是否支持HBV复制，形成包裹核酸的核心颗粒。结果 1成功构建在HEK293细胞内有效支持HBV复制的WtHBc与HBV1.1c-反式互补系统； 2HBc部位aa79-80缺失支持HBV制； 3HBc aa86-93缺失后功能缺； 4HBc N末插入肽后能较水平支持HBV制； 5HBc N末可为普有效位点，用A/B/C接连接，可支持片EGFP（238aa）RFP（225aa）、VSVG（511aa）。结论我们构建了有效HBc与HBV1.1c-反式互补统，统可检造HBc是否具有包裹核酸形成核心颗粒类病毒（VLPs）的功能。利用该系统，我们检测到HBc aa79-80及aa86-93缺失或替换后均存在功能缺陷，，这些序列可能与HBc的完整结构及功能有关。N末端可作为有效的改造位点，支持各种长度的外源蛋白融合，可长达511aa。
[Abstract]:Objective to investigate the modification site and form of HBc-based HBV core protein in order to form the core particles carrying cellular membrane penetrating peptide or ligand, which can be used as the basis for the modification of HBV infection model. Method. 1.1c- and all kinds of HBc modified plasmids were constructed by ligation-independent molecular cloning method (RLIC-). 2Cotransfection of various HBc recombined with HBV 1.1 c- into HEK293 cells. The nucleic acid nucleic acid core particles were extracted from the cells and detected by Southern blot to determine whether the HBc could support HBV replication and form nucleic acid nucleic acid core particles. Results. 1A WtHBc / HBV 1.1 c- trans complementary system was successfully constructed in HEK293 cells to effectively support HBV replication. 2HBC site aa79-80 deletion supported HBV system; 3functional deficiency after HBc aa86-93 deletion; 4HBc N terminal insertion peptide can support HBV system at a higher level; 5HBc N can be a common effective site, which can be connected with A / B / C, and can support EGFP238aa RFPN225aa / VSVG 511aAU. Conclusion We have constructed an effective HBc and HBV 1.1 c- trans complementary system, which can be used to detect whether HBc has the function of nucleic acid forming core granulovirus-like virus (HBc). Using this system, we can detect the functional defects of HBc aa79-80 and aa86-93 after deletion or replacement. These sequences may be related to the complete structure and function of HBc. The N terminal can be used as an effective modification site to support fusion of foreign proteins of various lengths and can be as long as 511aa.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62

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