AAV介导特异性表达siRNA的新型乙肝多表位疫苗的研究

发布时间：2018-04-10 20:56

本文选题：核酸疫苗 + 肝脏特异性　；参考：《华南理工大学》2012年硕士论文

【摘要】：据中国卫生部2010年最新统计，目前中国仍约有9300万人为无症状乙肝病毒携带者，乙型肝炎病毒的传染性比艾滋病毒强50至100倍，每年发病患者数在百万以上。乙肝免疫耐受是导致乙肝治疗失败的主要原因。临床治疗乙肝的药物存在高耐药、低耐受、不良反应等问题。传统的第二代疫苗本身的一些缺点（如部分接种者无免疫应答，出现副反应，对乙肝患者无效），及病毒本身不闭合等特点使得兼有预防和治疗作用的核酸疫苗成为热点。最新采用的RNAi技术治疗乙肝，虽然取得了一定的研究进展，但其组织特异性差，表达持续性弱，载体投递效率低，使得药物开发面临瓶颈。本研究采用肝脏特异性的二类启动子hAAT构建靶向HBV HBsAg基因的肝脏特异性siRNA表达单元，结合人工优选HBV表位构建多表位抗原基因HB，最终构建了AAV介导的特异性表达siRNA的新型乙肝多表位疫苗，为打破乙肝免疫耐受、消除基于RNAi技术药物的副作用以及提高疫苗的投递效率打下坚实的基础。首先通过重组PCR将肝脏特异性启动子hAAT、终止子U13’box、两个相同的增强子APOE及siHBsAg基因拼接构建成为肝脏特异性siHBsAg表达单元，将表达单元插入AAV-MCS质粒中，构建重组质粒pAAV-siHBsAg，ELISA检测siRNA的抑制效果。然后将HBV多表位抗原基因HB融合EGFP的蛋白基因通过分子克隆技术构建在pAAV-siHBsAg质粒中,得到pAAV-siHBsAg-HE，将重组质粒pAAV-siHBsAg-HE与包装质粒pAAV-RC和辅助质粒pHelper用磷酸钙法共转染AAV-293细胞制备重组病毒rAAV-siHBsAg-HE，并通过阴阳离子交换及超滤离心等纯化浓缩。构建的病毒rAAV-siHBsAg-HE感染HT1080细胞，通过观察绿色荧光，验证病毒的组装效果及HB-EGFP基因在AAV病毒载体中的表达，并且通过流式细胞术检测报告基因EGFP测定病毒滴度。再次使用ELISA检测基于腺相关病毒载体siRNA的抑制效果。最终结果是成功地构建了重组质粒pAAV-siHBsAg和pAAV-siHBsAg-HE。EGFP绿色荧光观察显示HB在rAAV中可以有效的表达。通过蛋白电泳检测及病毒电镜图片显示最终得到高纯度、高浓缩、高滴度的AAV病毒。ELISA检测表明构建的特异性表达siRNA的质粒和AAV病毒中均有抑制效果；AAV介导的特异性表达siRNA的新型乙肝多表位疫苗的成功构建和验证为进一步研究乙肝核酸疫苗提供了实验基础。
[Abstract]:According to the latest statistics from China's Ministry of Health in 2010, there are still about 93 million asymptomatic hepatitis B virus carriers in China. Hepatitis B virus is 50 to 100 times more infectious than HIV.Hepatitis B immune tolerance is the main cause of failure of hepatitis B treatment.Clinical treatment of hepatitis B drugs have high drug resistance, low tolerance, adverse reactions and other problems.Some disadvantages of the traditional second-generation vaccines (such as no immune response of some vaccinators, side effects, ineffective to hepatitis B patients, and the virus itself are not closed) make the nucleic acid vaccine with both preventive and therapeutic functions a hot spot.Although the latest RNAi technology has made some progress in the treatment of hepatitis B, it has poor tissue specificity, weak expression persistence and low delivery efficiency, which makes the drug development face the bottleneck.In this study, liver-specific siRNA expression units targeting HBV HBsAg gene were constructed by liver-specific second class promoter hAAT.In order to break the immune tolerance of hepatitis B virus, a novel hepatitis B polyepitope vaccine mediated by AAV was constructed by combining with the artificially selected HBV epitope gene HBs, and a novel hepatitis B polyepitope vaccine with specific expression of siRNA was constructed.Eliminating the side effects of drugs based on RNAi technology and improving the delivery efficiency of vaccines lay a solid foundation.Firstly, liver specific promoter hat, Terminator U13box, two identical enhancers APOE and siHBsAg genes were spliced into liver-specific siHBsAg expression units by recombinant PCR. The expression units were inserted into AAV-MCS plasmids.The inhibitory effect of recombinant plasmid pAAV-sibsAg on siRNA was detected by Elisa.Then the HBV polyepitope gene HB fused with EGFP protein gene was constructed into the pAAV-siHBsAg plasmid by molecular cloning.The recombinant plasmid pAAV-siHBsAg-HE, package plasmid pAAV-RC and auxiliary plasmid pHelper were co-transfected into AAV-293 cells by calcium phosphate method to prepare the recombinant virus rAAV-sibsAg-HEE. The recombinant virus was purified and concentrated by anion exchange and ultrafiltration centrifugation.The constructed virus rAAV-siHBsAg-HE was infected with HT1080 cells. By observing the green fluorescence, the viral assembly effect and the expression of HB-EGFP gene in the AAV virus vector were verified. The titer of the reporter gene EGFP was detected by flow cytometry.ELISA was used again to detect the inhibitory effect of adeno-associated virus vector siRNA.The final result was that the recombinant plasmid pAAV-siHBsAg and pAAV-siHBsAg-HE.EGFP were successfully constructed. The results showed that HB could be expressed effectively in rAAV.High purity and concentration were finally obtained by protein electrophoresis and electron microscope images of the virus.High titer detection of AAV virus. Elisa showed that the constructed plasmids expressing siRNA and AAV virus had inhibitory effect. The successful construction and validation of a novel hepatitis B multiepitope vaccine with specific siRNA expression mediated by AAV could be further studied.Liver nucleic acid vaccine provides experimental basis.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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