流感功能表位筛选与复合多表位核酸疫苗设计及免疫研究
发布时间：2018-11-23 08:41
【摘要】： 流感是由流感病毒引起的急性呼吸道传染病。几十年来,人流感的流行一直以A型H3/H1亚型以及B型流感为主,禽流感的流行以A型H5/H7/H9亚型为主,其流行已造成了巨大的损失。近年来,禽流感病毒的多个亚型(H5/H7/H9/H10)开始不断突破种间屏障感染人类,并可能造成大流行。当前,传统的流感疫苗已无法有效应对这一趋势,各国学者纷纷进行新型流感疫苗的开发以应对流感跨种传播及多亚型并存的现状。 本论文即拟对多亚型流感病毒复合多表位核酸疫苗进行研究。在搜集已发表流感功能表位的基础上,应用生物信息学理论对H1/H3/H5/H7/H9亚型的保护性抗原血凝素及神经氨酸酶进行了CTL、Th、B细胞表位的预测和筛选分析。设计组建了多表位功能基因盒,并进行了人工模拟优化,应用体外实验方式分析了预测的Th和B细胞功能表位抗原性。根据针对所属动物及预防目的的不同,我们构建了三种主导型H3/H1(针对人),H5/H3(针对人和猪),H5/H7(针对人和禽)的复合多表位核酸疫苗,通过RT-PCR及间接免疫荧光方法证实疫苗中各抗原组分可有效表达。最后,我们将所构建的复合多表位核酸疫苗进行了小鼠、猪、鸡的实验免疫研究,通过对ELISA及HI抗体水平、淋巴细胞刺激指数、IFNγ-ELISPOT和T淋巴细胞亚类数量、血清细胞因子含量等指标的综合分析,对不同复合多表位疫苗进行了免疫原性评价。结果显示,复合多表位疫苗组在针对主要抗原的抗体水平与不含表位组相当的情况下,表位相关抗原的抗体水平显示了一定的优势。表位相关抗原的淋巴细胞刺激指数显著升高,IFNγ-ELISPOT斑点数不同程度增加。复合多表位疫苗免疫组CD4~+T淋巴细胞数量和血清Th2类细胞因子含量显著增加。以上结果证实,复合多表位疫苗在诱导细胞及体液免疫方面均显示了优势,与各免疫对照组相比复合多表位DNA疫苗诱导的机体整体免疫水平最优。本研究对可同时预防多种亚型流感病毒,可对多种动物免疫保护,可提供机体长久免疫保护的跨种通用流感疫苗进行了有益尝试。
[Abstract]:Influenza is an acute respiratory infection caused by influenza virus. In recent decades, the prevalence of human influenza has been dominated by type A H3/H1 subtype and type B influenza, while the prevalence of avian influenza has been dominated by type A H5/H7/H9 subtype, which has caused great losses. In recent years, several subtypes (H5/H7/H9/H10) of avian influenza virus (AIV) begin to break through interspecific barriers and infect humans, which may cause a pandemic. At present, the traditional influenza vaccine has been unable to effectively cope with this trend. Scholars from all over the world have developed new influenza vaccines to cope with the interspecies transmission and coexistence of multiple subtypes of influenza. In this paper, we intend to study the multiple subtype influenza virus complex polyepitope nucleic acid vaccine. Based on the collection of published functional epitopes of influenza, CTL,Th,B cell epitopes were predicted and screened by bioinformatics theory for the protective antigens hemagglutinin and neuraminidase of H1/H3/H5/H7/H9 subtypes. The multiepitope functional gene cassette was designed and optimized by artificial simulation. The predicted antigenicity of Th and B cell functional epitopes was analyzed by means of in vitro experiments. According to the difference between animals and prevention purposes, we have constructed three kinds of complex polyepitope nucleic acid vaccines of H3/H1 (for human), H5/H3 (for human and pig), H5/H7 (for human and poultry). RT-PCR and indirect immunofluorescence assay were used to confirm the effective expression of each antigen component in the vaccine. Finally, we carried out the experimental immunological study of the complex polyepitope nucleic acid vaccine in mice, pigs and chickens. The levels of ELISA and HI antibodies, lymphocyte stimulating index (LSI), IFN 纬-ELISPOT and the number of T lymphocyte subclasses were measured. The immunogenicity of different multiepitope vaccines was evaluated by comprehensive analysis of serum cytokines. The results showed that the antibody level of epitope associated antigen in the composite multiepitope vaccine group was similar to that in the non-epitope group, and the antibody level of epitope associated antigen showed some advantages. The lymphocyte stimulating index of epitope associated antigen increased significantly, and the number of IFN 纬-ELISPOT spots increased in varying degrees. The number of CD4~ T lymphocytes and the content of serum Th2 cytokines were significantly increased in multiple epitope vaccine immunized group. The above results confirmed that the compound polyepitope vaccine had the advantages in inducing cellular and humoral immunity, and the overall immune level induced by the compound polyepitope DNA vaccine was the best compared with that of the control group. This study is a useful attempt to prevent multiple subtypes of influenza virus simultaneously, to protect animal immunity, and to provide long term immunity protection for a variety of general influenza vaccines.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392
本文编号：2350940
[Abstract]:Influenza is an acute respiratory infection caused by influenza virus. In recent decades, the prevalence of human influenza has been dominated by type A H3/H1 subtype and type B influenza, while the prevalence of avian influenza has been dominated by type A H5/H7/H9 subtype, which has caused great losses. In recent years, several subtypes (H5/H7/H9/H10) of avian influenza virus (AIV) begin to break through interspecific barriers and infect humans, which may cause a pandemic. At present, the traditional influenza vaccine has been unable to effectively cope with this trend. Scholars from all over the world have developed new influenza vaccines to cope with the interspecies transmission and coexistence of multiple subtypes of influenza. In this paper, we intend to study the multiple subtype influenza virus complex polyepitope nucleic acid vaccine. Based on the collection of published functional epitopes of influenza, CTL,Th,B cell epitopes were predicted and screened by bioinformatics theory for the protective antigens hemagglutinin and neuraminidase of H1/H3/H5/H7/H9 subtypes. The multiepitope functional gene cassette was designed and optimized by artificial simulation. The predicted antigenicity of Th and B cell functional epitopes was analyzed by means of in vitro experiments. According to the difference between animals and prevention purposes, we have constructed three kinds of complex polyepitope nucleic acid vaccines of H3/H1 (for human), H5/H3 (for human and pig), H5/H7 (for human and poultry). RT-PCR and indirect immunofluorescence assay were used to confirm the effective expression of each antigen component in the vaccine. Finally, we carried out the experimental immunological study of the complex polyepitope nucleic acid vaccine in mice, pigs and chickens. The levels of ELISA and HI antibodies, lymphocyte stimulating index (LSI), IFN 纬-ELISPOT and the number of T lymphocyte subclasses were measured. The immunogenicity of different multiepitope vaccines was evaluated by comprehensive analysis of serum cytokines. The results showed that the antibody level of epitope associated antigen in the composite multiepitope vaccine group was similar to that in the non-epitope group, and the antibody level of epitope associated antigen showed some advantages. The lymphocyte stimulating index of epitope associated antigen increased significantly, and the number of IFN 纬-ELISPOT spots increased in varying degrees. The number of CD4~ T lymphocytes and the content of serum Th2 cytokines were significantly increased in multiple epitope vaccine immunized group. The above results confirmed that the compound polyepitope vaccine had the advantages in inducing cellular and humoral immunity, and the overall immune level induced by the compound polyepitope DNA vaccine was the best compared with that of the control group. This study is a useful attempt to prevent multiple subtypes of influenza virus simultaneously, to protect animal immunity, and to provide long term immunity protection for a variety of general influenza vaccines.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392
【引证文献】
相关期刊论文 前1条
1 谭磊;鲁会军;张丹;王开艳;田明尧;刘存霞;刘燕瑜;胡博;金宁一;;季节性和大流行性H1N1流感血凝素DNA疫苗电穿孔免疫小鼠及攻毒保护实验[J];中国科学:生命科学;2011年02期
相关博士学位论文 前1条
1 谭磊;流感H3/H1亚型多表位核酸疫苗和重组腺病毒疫苗构建及实验免疫研究[D];吉林大学;2011年
相关硕士学位论文 前1条
1 王开艳;H3/H1亚型流感核酸疫苗的构建及小鼠免疫试验[D];延边大学;2010年
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