基于流感病毒血凝素抗原表位的核酸疫苗研究

发布时间：2018-09-13 05:56

【摘要】：流感一直是人类社会的一个巨大威胁。应对流感,最好的办法就是接种疫苗。相对于传统的灭活疫苗和减毒活疫苗,新出现的核酸疫苗基于它的众多优点,很有希望成为新一代的流感疫苗。由于流感病毒表面抗原血凝素(HA)是流感病毒核酸疫苗最好的靶点,而针对血凝素抗原表位产生的抗体是主要的具保护性的中和抗体,因此对基于血凝素抗原表位的核酸疫苗进行的研究具有一定的探索性意义。本文在小鼠模型中探讨了血凝素单个抗原表位的改变对于核酸疫苗保护效果的影响；并尝试构建基于血凝素表位的具有交叉保护效果的核酸疫苗。本论文包括以下几个部分：(一)流感病毒,流感病毒疫苗和流感病毒血凝素的简介；(二)含甲型H1N1流感病毒血凝素单个抗原表位核酸疫苗的构建及其免疫小鼠后的保护效果比较研究；(三)含流感病毒血凝素多个抗原表位核酸疫苗的构建及其免疫小鼠后的交叉保护效果研究。 (一)流感病毒,流感病毒疫苗和流感病毒血凝素的简介：以综述的形式简单介绍了流感病毒及其变异性；流感病毒疫苗的分类和特点；流感病毒血凝素的特性。 (二)含甲型H1N1流感病毒血凝素单个抗原表位的核酸疫苗的构建及其免疫小鼠后的保护效果比较研究：试验中构建了多个以A/PR/8流感病毒HA1为骨架,含不同甲型H1N1流感病毒单个表位的核酸疫苗。分别将各疫苗30微克使用肌肉注射后辅以电击的方式免疫BALB/c雌性小鼠,一次免疫三周后或两次免疫一周后用致死量(20×LD50)甲型H1N1流感病毒攻击小鼠,通过观察小鼠的体重丢失率和存活率来比较评价各个疫苗的保护作用。结果显示,含甲型H1N1流感病毒血凝素单个抗原表位的核酸疫苗能够提供小鼠抗致死量流感病毒攻击的能力,一次免疫后可以提供部分保护,两次免疫后基本能达到完全保护。 (三)含流感病毒血凝素多个抗原表位核酸疫苗的构建及其免疫小鼠后的交叉保护效果研究：我们在试验中构建了以甲型H1N1流感病毒HA1为骨架,含两个A/PR/8流感病毒表位的核酸疫苗。分别用含甲型H1N1流感病毒全长HA和A/PR/8流感病毒全长HA的核酸疫苗为对照。将各疫苗都用30微克间隔三周两次电击免疫BALB/c雌性小鼠,两免二周后分别用致死量(20×LD50)甲型H1N1流感病毒和致死量(20×LD50)A/PR/8流感病毒攻击小鼠,通过观察小鼠的体重丢失率和存活率来评价该疫苗的交叉保护作用。结果显示,两次免疫后,该核酸疫苗对甲型H1N1流感病毒和A/PR/8流感病毒的攻击都能提供完全的保护,达到了和含相应病毒株全长HA核酸疫苗一样的效果,而对照组基本没有或只有很小的保护效果。因此我们认为,基于流感病毒血凝素多个抗原表位的核酸疫苗方案是可行的,可以到达对于多个病毒株的交叉保护效果。
[Abstract]:Influenza has always been a great threat to human society. The best way to deal with influenza is to get vaccinated. Compared with the traditional inactivated vaccine and attenuated live vaccine, the new nucleic acid vaccine is expected to become a new generation of influenza vaccine because of its many advantages. Because influenza virus surface antigen hemagglutinin (HA) is the best target for influenza virus nucleic acid vaccine, antibodies against hemagglutinin epitopes are the main protective neutralizing antibodies. Therefore, the research of nucleic acid vaccine based on hemagglutinin epitope has some exploratory significance. In this paper, we studied the effect of the change of single antigen epitope of hemagglutinin on the protective effect of nucleic acid vaccine in mice, and tried to construct a cross-protective nucleic acid vaccine based on hemagglutinin epitope. This paper includes the following parts: (1) Influenza virus, influenza virus vaccine and influenza virus hemagglutinin; (2) Construction of single epitope nucleic acid vaccine containing hemagglutinin of influenza A H1N1 virus and comparative study on its protective effect after immunizing mice; (3) Construction of multiantigen epitope nucleic acid vaccine containing hemagglutinin of influenza virus and its cross-protective effect after immunizing mice. Introduction of influenza virus, influenza virus vaccine and influenza virus hemagglutinin: a brief introduction to influenza virus and its variability; classification and characteristics of influenza virus vaccine; and characteristics of influenza virus hemagglutinin (2) Construction of nucleic acid vaccine containing hemagglutinin epitope of influenza A H1N1 virus and comparative study of its protective effect after immunizing mice: in the experiment, several A/PR/8 influenza virus HA1 was constructed as skeleton. Nucleic acid vaccine containing a single epitope of different influenza A H1N1 viruses. 30 micrograms of each vaccine were injected intramuscularly to immunize BALB/c female mice with electric shock. The mice were immunized three weeks later or two weeks later with a lethal dose of (20 脳 LD50) H1N1 influenza virus. The protective effects of each vaccine were evaluated by observing the weight loss rate and survival rate of mice. The results showed that the nucleic acid vaccine containing hemagglutinin epitopes of influenza A H1N1 virus could provide the ability of resisting lethal influenza virus attack in mice. (3) Construction of multi-antigen epitope nucleic acid vaccine containing hemagglutinin of influenza virus and its cross-protective effect after immunizing mice: we constructed a H1N1 influenza virus HA1 skeleton in our experiment. Nucleic acid vaccine containing two A/PR/8 influenza epitopes. Nucleic acid vaccines containing full-length HA of A H1N1 influenza virus and full length HA of A/PR/8 influenza virus were used as control group. BALB/c female mice were immunized with 30 micrograms of each vaccine twice every three weeks. After two weeks of immunization, the mice were attacked with 20 脳 LD50 H1N1 influenza virus and 20 脳 LD50 A/PR/8 influenza virus, respectively. The cross-protection of the vaccine was evaluated by observing the weight loss rate and survival rate of the mice. The results showed that after twice immunization, the nucleic acid vaccine could provide complete protection against influenza A H1N1 virus and A/PR/8 influenza virus, and achieved the same effect as the full-length HA nucleic acid vaccine containing the corresponding virus strain. The control group had little or no protective effect. Therefore, we believe that the nucleic acid vaccine based on multiple antigen epitopes of influenza virus hemagglutinin is feasible and can achieve cross-protection for multiple virus strains.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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