整合siRNA技术的结核核酸疫苗AAV病毒载体构建与浓缩纯化

发布时间：2018-07-03 12:47

本文选题：结核疫苗 + 腺相关病毒　；参考：《华南理工大学》2011年硕士论文

【摘要】：结核病可累及全身多个脏器,其中肺结核最为常见。目前我国是世界上22个结核病高负担国家之一,我国三分之一左右的人口已感染了结核杆菌,受感染人数超过4亿。我国现有肺结核病人约500万。如果不采取有效的控制措施,在未来的10年,我国可能有近5000万的感染者发生结核病。对结核病的防治传统的方法为接种卡介苗,但是缺陷大,反应差异明显,不能用于免疫缺陷的个体,同时为以后的检测带来诸多不便。研制更有效的结核疫苗具有深远的意义。目的:以最新腺相关病毒载体系统,构建携带肺结核融合抗原(Ag85b-ESAT6)并整合siRNA(si-MCL-1)表达单位的二重表达新型结核核酸疫苗。包装出含有目标基因的重组腺相关病毒颗粒。分别构建rAAV-EGFP与rAAV-siEGFP-EGFP腺相关病毒,包装并通过病毒介导目标基因在宿主细胞进行表达,验证病毒载体表达水平。通过超滤浓缩纯化rAAV病毒颗粒,提高病毒载体感染效率。方法:利用genetool软件工具分析AAV-Helper-free系统中pAAV-MCS质粒中的全部酶切位点,在不影响病毒包装的前提下,确定酶切位点MluI作为siRNA表达框插入点。通过PCR方法扩增融合抗原Ag85b-ESAT6与si-MCL-1(包括H1启动子),si-EGFP(包括H1启动子),将目的基因和表达框插入pAAV-MCS多克隆位点及相应酶切位点,构建并包装rAAV-si-MCL-1-pVAE,rAAV-siEGFP-EGFP,rAAV-EGFP。其中pAAV-EGFP表达质粒为实验室已有资源。通过磷酸钙共沉淀法将重组质粒与包装辅助质粒pAAV-Helper,pAAV-RC共转染AAV-293细胞制备重组病毒。重组病毒经超滤浓缩,通过荧光显微镜以及流式细胞仪检测表达效果。结果:重组子pAAV-si-MCL-1-pVAE ,pAAV-siEGFP-EGFP通过PCR鉴定和测序证实构建成功。病毒感染HT1080在荧光显微镜和流式细胞仪中检测到绿色荧光并存在差异,表明重组病毒包装成功,siRNA表达单位正常工作不影响病毒包装和蛋白表达。结论:成功包装出具有侵染性和正常表达外源目的基因的重组腺相关病毒rAAV-si-MCL-1-pVAE,rAAV-siEGFP-EGFP,rAAV-EGFP。浓缩和纯化后的rAAV病毒颗粒感染HT1080效果更明显。本研究为研制优于BCG的疫苗提供了新思路,具有深远的意义。
[Abstract]:Tuberculosis can involve multiple organs throughout the body, of which tuberculosis is the most common. At present, China is one of the 22 countries with high TB burden in the world. About 1/3 people in our country have been infected with Mycobacterium tuberculosis, and the number of infected people is more than 400 million. There are about 5 million tuberculosis patients in China. If effective control measures are not taken, there may be nearly 50 million cases of tuberculosis in China in the next 10 years. The traditional method to prevent and cure tuberculosis is to vaccinate BCG vaccine, but the defect is big, the reaction difference is obvious, can't be used in the individual of the immune deficiency, at the same time, bring many inconvenience for the later examination at the same time. It is of great significance to develop a more effective TB vaccine. Aim: to construct a novel recombinant tuberculosis nucleic acid vaccine carrying ag85b-ESAT6 and siRNA (si-MCL-1) expression unit by using the latest adeno-associated virus vector system. The recombinant adeno-associated virus particles containing the target gene were packaged. RAAV-EGFP and rAAV-siEGFP-EGFP adeno-associated viruses were constructed, and the target genes were expressed in host cells by virus mediated expression. RAAV virus particles were concentrated and purified by ultrafiltration to improve the efficiency of viral vector infection. Methods: the restriction sites of pAAV-MCS plasmid in AAV-Helper-free system were analyzed by genetool software, and the restriction site MluI was determined as the insertion point of siRNA expression frame without affecting virus packaging. The fusion antigen Ag85b-ESAT6 and si-MCL-1 (including H1 promoter) were amplified by PCR. The target gene and expression frame were inserted into the polyclonal site of pAAV-MCS and the corresponding restriction site, and the rAAV-siEGFP-EGFP pAAV-siEGFP-EGFP was constructed and packaged by PCR. Among them, pAAV-EGFP expression plasmid is a resource of laboratory. The recombinant virus was prepared by co-transfection of recombinant plasmid and pAAV-Helperon pAAV-RC into AAV-293 cells by calcium superphosphate coprecipitation. The recombinant virus was concentrated by ultrafiltration and detected by fluorescence microscope and flow cytometry. Results: the recombinant pAAV-si-MCL-1-pVAE pAAV-siEGFP-EGFP was successfully constructed by PCR and sequencing. Green fluorescence was detected by fluorescence microscope and flow cytometry in HT1080 infected by the virus, which indicated that the successful packaging of recombinant virus did not affect the viral packaging and protein expression. Conclusion: the recombinant adeno-associated virus rAAV-si-MCL-1-pVAEN-rAAV-siEGFP-EGFPN rAAV-EGFPhas been successfully packaged with infectious and normal expression of exogenous target gene. The concentrated and purified rAAV particles infected with HT1080 were more effective. This study provides a new idea for the development of a vaccine better than BCG, and has far-reaching significance.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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