人Tollip基因真核表达载体的构建及其抗HBV的体外研究

发布时间：2018-06-04 15:04

本文选题：HBV + Tollip　；参考：《重庆医科大学》2012年硕士论文

【摘要】：乙型肝炎病毒（Hepatitis B Virus，HBV）的感染在中国高发，其持续感染易导致肝硬化和肝癌，严重危害人类健康，所以防治HBV感染成为我国传染病研究的热点。乙肝疫苗对未感染者具有明显保护作用，但是，HBV感染尤其是慢性感染的治疗仍然面临很大的困难。目前，核苷类药物和干扰素被广泛用于抗HBV治疗，但缺乏切实有效的长期应答效应以及个体耐药等因素制约了疗效，亟需寻找新的治疗靶点和药物。Toll样受体（Toll like receptors，TLRs）作为一种重要的病原生物模式识别受体，能够识别HBV等病毒。TLR/IL-1R信号转导通路，在炎症反应和机体天然免疫中起着举足轻重的作用，该通路中的关键信号分子可能成为抗HBV的新靶点。Toll作用蛋白(Toll-interacting protein，Tollip)是TLR/IL-1R信号转导通路中的一个关键接头蛋白，它对HBV的感染有何影响是一个值得探讨的问题。本文通过构建含HA标签蛋白的Tollip真核表达载体pCMV-HA-Tollip，并成功转染人肝癌细胞株HepG2.2.15，通过观察其对HBV抗原的影响，分析TLR/IL-1R信号转导途径中重要分子的表达,以期初步阐明Tollip在抗HBV感染中的作用，为乙肝的治疗提供新的理论依据和治疗靶点。具体内容如下：目的：构建含HA标签的重组表达质粒pCMV-HA-Tollip，转染HepG2.2.15细胞，检测其对HBV抗原分泌的影响并探讨其机制。方法：1.构建真核表达载体pCMV-HA-Tollip双酶切及DNA测序鉴定。2.采用脂质体转染法，将质粒pCMV-HA-Tollip转染HepG2.2.15细胞。3.ELISA法检测HBsAg和HBeAg的表达。4.Western Blot法检测AKT、p-AKT、NF-κB的差异表达。结果：1.成功构建了真核表达载体pCMV-HA-Tollip。2.质粒成功转染HepG2.2.15细胞48h后，3μg空质粒与3μg pCMV-HA-Tollip共转染组、6μg pCMV-HA-Tollip转染组中HepG2.2.15细胞的HBsAg和HBeAg分泌量明显低于6μg空质粒转染对照组，对HBsAg的抑制率分别为24%和41%(p＜0.01)，对HBeAg的抑制率分别为13%和31%(p＜0.01)；与6μg空质粒转染组相比，3μg空质粒与3μg pCMV-HA-Tollip共转染组和6μg pCMV-HA-Tollip转染组中NF-κB、p-AKT的表达明显降低(p＜0.01)，而AKT的表达没有明显的变化。结论：1.成功构建pCMV-HA-Tollip重组真核表达质粒。2.pCMV-HA-Tollip重组质粒在HepG2.2.15细胞中能有效抑制HBsAg和HBeAg的分泌，，其机制可能与抑制AKT的磷酸化和下调NF-κB的表达有关。
[Abstract]:Hepatitis B virus (HBV) infection is highly prevalent in China, and its persistent infection is liable to lead to cirrhosis and liver cancer, which seriously endangers human health. Therefore, the prevention and treatment of HBV infection has become a hot topic in infectious diseases research in China. Hepatitis B vaccine has obvious protective effect on uninfected patients, but the treatment of HBV infection, especially chronic infection, still faces great difficulties. At present, nucleoside drugs and interferon are widely used in the treatment of HBV, but lack of effective long-term response effect and individual drug resistance restrict the efficacy. There is an urgent need to find new therapeutic targets and drugs. Toll like receptor TLRs), as an important pathogenetic biologic pattern recognition receptor, can recognize the signal transduction pathway of virus. TLR / IL-1R, such as HBV, and play an important role in inflammatory response and innate immunity. The key signal molecule in this pathway may be a new target of anti-HBV. Toll-interacting protein Tollipase is a key junction protein in TLR/IL-1R signal transduction pathway. The influence of Toll-interacting protein on HBV infection is a problem to be discussed. In this paper, Tollip eukaryotic expression vector pCMV-HA-Tollip-containing HA label protein was constructed and transfected into HepG2.2.15. the expression of important molecules in TLR/IL-1R signal transduction pathway was analyzed by observing its effect on HBV antigen. The aim of this study was to clarify the role of Tollip in anti-HBV infection and to provide new theoretical basis and therapeutic target for the treatment of hepatitis B. The details are as follows: Aim: to construct a recombinant expression plasmid pCMV-HA-Tollip-containing HA label and transfect it into HepG2.2.15 cells to investigate the effect of pCMV-HA-Tollipase on the secretion of HBV antigen and its mechanism. Method 1: 1. Construction of eukaryotic expression vector pCMV-HA-Tollip double digestion and DNA sequencing identification. 2. 2. The expression of HBsAg and HBeAg was detected by Elisa in HepG2.2.15 cells transfected with plasmid pCMV-HA-Tollip by liposome transfection. 4. Western Blot method was used to detect the differential expression of AKTP-AKTN NF- 魏 B in HepG2.2.15 cells. The result is 1: 1. The eukaryotic expression vector pCMV-HA-Tollip.2was successfully constructed. The HBsAg and HBeAg secretion of HepG2.2.15 cells in the 3 渭 g pCMV-HA-Tollip co-transfected group was significantly lower than that in the 6 渭 g empty plasmid transfected control group after 48 hours of successful transfection of the plasmid into HepG2.2.15 cells. The inhibition rates of HBsAg and HBeAg were 24% and 41%, respectively, and those of HBeAg were 13% and 31%, respectively, and the expression of NF- 魏 Bp-AKT was significantly lower than that of 6 渭 g empty plasmid transfection group and 3 渭 g pCMV-HA-Tollip co-transfection group and 6 渭 g pCMV-HA-Tollip group, but the expression of AKT had no significant change. Conclusion 1. The successful construction of pCMV-HA-Tollip recombinant eukaryotic expression plasmid. 2. PCMV-HA-Tollip recombinant plasmid can effectively inhibit the secretion of HBsAg and HBeAg in HepG2.2.15 cells. The mechanism may be related to the inhibition of AKT phosphorylation and down-regulation of NF- 魏 B expression.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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