噬菌体展示候选甲型流感病毒和乙型流感病毒联合疫苗的构建及免疫原性研究
发布时间：2018-10-16 17:08
【摘要】： 流行性感冒是一种非常严重的全球性的呼吸道传染病。在流感流行期间，全球有10—20％的人口感染流感病毒，每年造成三百万至五百万严重病例和250，000至500，000例死亡。对流感的预防尚无非常有效的方法，接种疫苗是目前最有效的途径。血凝素(HA)和神经氨酸(NA)是存在于病毒表面易感的大型糖蛋白，且具有强的免疫原性，是非常好的疫苗抗原。现在市场上的疫苗都将血凝素(hemagglutinin，HA)和神经氨酸(neuraminidase，NA)作为主要或唯一病毒抗原。血凝素(HA)和神经氨酸(NA)基因的改变和重排，是通过疫苗接种控制流感流行的主要障碍。因此，流感病毒能够重复感染并使得每年需要新的疫苗进行免疫接种。开发基于流感病毒不变区域，能产生广谱的交叉免疫反应的普适性流感疫苗是迫切需要的。 由病毒基因编码的M2蛋白是一个四聚体离子通道，在病毒颗粒中少量表达或不表达，但却在被病毒感染的细胞表面大量表达。M2蛋白膜外区(M2e)由24个氨基酸残基组成，并且不发生基因漂移和变异。自1918年西班牙流感大流行至今，虽然其间发生了无数次的流感流行和三次全球性大流行，M2e序列基本保持不变。研究表明，不同构造形式的基于M2e的疫苗能够提供保护性免疫。乙型流感病毒的BM2蛋白由109个氨基酸残基组成，是乙型流感病毒编码的血凝素(HA)、神经氨酸(NA)和NB糖蛋白之外的第四个膜内在蛋白。BM2具有离子通道活性，荧光显微镜显示BM2表达于为感染细胞表面。虽然BM2在乙型流感病毒生活周期中有着自己特有的功能，但是BM2还是被认为具有和M2蛋白在甲型流感病毒中相似的功能，可能能作为通用乙型流感病毒的候选表位。 流感病毒研究中常用的是小鼠动物模型不能在小鼠间传播，而豚鼠不但能对未经适应性改变的流感病毒敏感，流感病毒也能在豚鼠中以飞沫传播，因此豚鼠可以作为流感病毒传染研究的新动物模型。噬菌体疫苗是以噬菌体作为疫苗载体，将特异表位展示在噬菌体颗粒表面，构建成不同表位特异的噬菌体疫苗，具有病毒样颗粒特性，能引发强的针对表位的特异性免疫应答。鼻腔免疫是近年发展起来新的粘膜免疫途径，它通过抗原在鼻粘膜的释放，诱导系统和粘膜免疫应答。研究表明，由T细胞、B细胞、树突细胞以及包括M细胞的隐窝吸收上皮细胞组成的鼻粘膜相关淋巴组织(nose associated lymphoid tissue，NALT)，是鼻腔免疫的诱导部位。壳聚糖是一个新型的缓控释疫苗呈递系统载体，能安全而有效地促进鼻腔吸收微球疫苗。 本文利用基因重组将M2eBM2多表位基因片断与T7 10B衣壳蛋白基因融合构建噬菌体候选疫苗，PCR鉴定为阳性的噬菌体候选疫苗扩增后用甲醛灭活。用10~(10)pfu剂量的噬菌体微球联合流感候选疫苗经鼻腔免疫豚鼠，并用10~3pfu的H3N2、H1N1和B型流感病毒分别对经候选疫苗免疫和未经候选疫苗免疫的豚鼠进行攻击，分别检测上呼吸道(upstream respiratory tract，URT)和肺部流感病毒滴度，，对比研究候选疫苗对病毒感染的保护作用。 ELISA检测血清IgG水平发现，噬菌体微球联合流感候选疫苗具有特异性免疫原性，能诱导产生针对M2e和BM2的特异性抗体。对病毒感染的豚鼠呼吸道内的病毒滴度检测表明，噬菌体微球联合流感候选疫苗对豚鼠的全呼吸道具有抗H3N2和H1N1的保护作用；对乙型流感病毒感染的豚鼠上呼吸道候选疫苗无保护效果，肺部有一定的保护效果。 因此，(1)构建的噬菌体微球联合流感候选疫苗经鼻腔免疫能有效诱导特异性体液免疫，产生特异性抗体(M2eBM2)；(2)候选疫苗诱导的M2e抗体对感染H3N2和H1N1的豚鼠的全呼吸道有保护作用；(3)候选疫苗诱导的BM2抗体对感染乙流感病毒的豚鼠肺部有保护作用而对上呼吸道无保护效果。
[Abstract]:Influenza is a very serious global respiratory infectious disease. During the pandemic, 10 to 20% of the world's population is infected with influenza viruses, causing three to five million serious cases per year and 250,000 to 500,000 deaths each year. There is no effective way of preventing influenza, and vaccination is the most effective way. Hemagglutinin (HA) and neuramine (NA) are large glycoproteins present on the surface of the virus and have strong immunogenicity and are very good vaccine antigens. Vaccines in the market now use haemagglutinin (HA) and neuraminase (NA) as primary or only viral antigens. Changes and rearrangements of hemagglutinin (HA) and neuramine (NA) genes are major obstacles to the control of influenza epidemics by vaccination. Thus, the influenza virus is able to repeat infection and make it necessary to immunize each year with a new vaccine. It is urgent to develop a universal influenza vaccine based on influenza virus invariant regions, which can produce a broad spectrum of cross-immune responses. The M2 protein encoded by the viral gene is a tetramer ion channel that is expressed or not expressed in a small amount in the viral particles but is large in the surface of the infected cell The M2 protein film outer region (M2e) consists of 24 amino acid residues and no gene drift occurs and variation. Since the 1918 Spanish flu pandemic, the M2e sequence is basically guaranteed, although numerous influenza epidemics and three global epidemics have occurred between them. Invariant. Studies have shown that M2e-based vaccines in different configurations can provide protection The BM2 protein of influenza B is composed of 109 amino acid residues, and is the fourth membrane outside the hemagglutinin (HA), neuramine (NA) and NB glycoprotein encoded by influenza B virus. BM2 has an ion channel activity and the fluorescence microscope shows that BM2 is expressed as a fine infection Although BM2 has its own specific function in the life cycle of influenza B, BM2 is considered to have a similar function to the M2 protein in influenza A virus and can be used as a general influenza B virus. It is common in the study of influenza viruses that mouse models cannot be propagated between mice, and guinea pigs can not only be sensitive to an unadaptable influenza virus, but also in guinea pigs, so that guinea pigs can be infected as influenza viruses. According to the new animal model, phage vaccine is used as a vaccine vector, the specific table bit is displayed on the surface of the phage particles, the specific phage vaccine is constructed into different table bits, Specific immune response. Nasal immunization is a new way of mucosal immune development in recent years. It induces the release and induction of nasal mucosa by antigen. It is indicated that the nasal mucosa-associated lymphoid tissue (NALT) composed of T cells, B cells, dendritic cells, and crypt-absorbing epithelial cells including M cells, is the nasal cavity. Chitosan is a new kind of sustained-release vaccine presenting system carrier, which can promote nose safely and effectively. In this paper, a phage candidate vaccine was constructed by fusion of MineBM2 multi-table gene fragment with T7 10B capsid protein gene by gene recombination, and PCR was identified as a positive phage candidate. Using 10 ~ (10) Vu doses of phage micro-ball combined with influenza candidate vaccine to immunize guinea pig via nasal cavity, and using H3N2, H1N1 and Btype influenza viruses of 10 ~ 32.5u to attack guinea pigs immunized with candidate vaccine and uncandidate vaccine, respectively, detect upper respiratory tract (upstream response), respectively. Respiratory tract, URT, and lung influenza virus drops, comparative study candidate Detection of serum IgG level by ELISA shows that phage microspheres combined with influenza candidate vaccine have specific immunogenicity and can induce production. Specific antibodies against M2e and BM2. Detection of the virus drop in the respiratory tract of guinea pigs infected by the virus showed that phage microspheres combined with influenza candidate vaccines had a protective effect on the whole respiratory tract of guinea pigs against H3N2 and H1N1; influenza B virus infection Guinea pig upper respiratory tract candidate vaccine free Therefore, (1) the constructed phage micro-ball combined influenza candidate vaccine can effectively induce specific humoral immunity through nasal immunization and generate specific antibody (M2eBM2); (2) the M2e antibody induced by the candidate vaccine is infected with H3N. 2 and 1 (H1N1) guinea pigs have a protective effect on the whole respiratory tract; (3) a candidate vaccine-induced BM2 antibody is associated with a dolphin infected with an influenza B virus
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
本文编号：2275066
[Abstract]:Influenza is a very serious global respiratory infectious disease. During the pandemic, 10 to 20% of the world's population is infected with influenza viruses, causing three to five million serious cases per year and 250,000 to 500,000 deaths each year. There is no effective way of preventing influenza, and vaccination is the most effective way. Hemagglutinin (HA) and neuramine (NA) are large glycoproteins present on the surface of the virus and have strong immunogenicity and are very good vaccine antigens. Vaccines in the market now use haemagglutinin (HA) and neuraminase (NA) as primary or only viral antigens. Changes and rearrangements of hemagglutinin (HA) and neuramine (NA) genes are major obstacles to the control of influenza epidemics by vaccination. Thus, the influenza virus is able to repeat infection and make it necessary to immunize each year with a new vaccine. It is urgent to develop a universal influenza vaccine based on influenza virus invariant regions, which can produce a broad spectrum of cross-immune responses. The M2 protein encoded by the viral gene is a tetramer ion channel that is expressed or not expressed in a small amount in the viral particles but is large in the surface of the infected cell The M2 protein film outer region (M2e) consists of 24 amino acid residues and no gene drift occurs and variation. Since the 1918 Spanish flu pandemic, the M2e sequence is basically guaranteed, although numerous influenza epidemics and three global epidemics have occurred between them. Invariant. Studies have shown that M2e-based vaccines in different configurations can provide protection The BM2 protein of influenza B is composed of 109 amino acid residues, and is the fourth membrane outside the hemagglutinin (HA), neuramine (NA) and NB glycoprotein encoded by influenza B virus. BM2 has an ion channel activity and the fluorescence microscope shows that BM2 is expressed as a fine infection Although BM2 has its own specific function in the life cycle of influenza B, BM2 is considered to have a similar function to the M2 protein in influenza A virus and can be used as a general influenza B virus. It is common in the study of influenza viruses that mouse models cannot be propagated between mice, and guinea pigs can not only be sensitive to an unadaptable influenza virus, but also in guinea pigs, so that guinea pigs can be infected as influenza viruses. According to the new animal model, phage vaccine is used as a vaccine vector, the specific table bit is displayed on the surface of the phage particles, the specific phage vaccine is constructed into different table bits, Specific immune response. Nasal immunization is a new way of mucosal immune development in recent years. It induces the release and induction of nasal mucosa by antigen. It is indicated that the nasal mucosa-associated lymphoid tissue (NALT) composed of T cells, B cells, dendritic cells, and crypt-absorbing epithelial cells including M cells, is the nasal cavity. Chitosan is a new kind of sustained-release vaccine presenting system carrier, which can promote nose safely and effectively. In this paper, a phage candidate vaccine was constructed by fusion of MineBM2 multi-table gene fragment with T7 10B capsid protein gene by gene recombination, and PCR was identified as a positive phage candidate. Using 10 ~ (10) Vu doses of phage micro-ball combined with influenza candidate vaccine to immunize guinea pig via nasal cavity, and using H3N2, H1N1 and Btype influenza viruses of 10 ~ 32.5u to attack guinea pigs immunized with candidate vaccine and uncandidate vaccine, respectively, detect upper respiratory tract (upstream response), respectively. Respiratory tract, URT, and lung influenza virus drops, comparative study candidate Detection of serum IgG level by ELISA shows that phage microspheres combined with influenza candidate vaccine have specific immunogenicity and can induce production. Specific antibodies against M2e and BM2. Detection of the virus drop in the respiratory tract of guinea pigs infected by the virus showed that phage microspheres combined with influenza candidate vaccines had a protective effect on the whole respiratory tract of guinea pigs against H3N2 and H1N1; influenza B virus infection Guinea pig upper respiratory tract candidate vaccine free Therefore, (1) the constructed phage micro-ball combined influenza candidate vaccine can effectively induce specific humoral immunity through nasal immunization and generate specific antibody (M2eBM2); (2) the M2e antibody induced by the candidate vaccine is infected with H3N. 2 and 1 (H1N1) guinea pigs have a protective effect on the whole respiratory tract; (3) a candidate vaccine-induced BM2 antibody is associated with a dolphin infected with an influenza B virus
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392
【引证文献】
相关期刊论文 前2条
1 汤洋;王海漩;乌美妮;胡凝珠;胡云章;;流感病毒感染豚鼠模型的初步建立[J];中国生物制品学杂志;2011年03期
2 王海漩;乌美妮;汤洋;李建芳;李彦涵;胡云章;赵蕊蕊;沈霏;段志青;胡凝珠;;广谱流感噬菌体微球疫苗对豚鼠的免疫保护作用[J];中国生物制品学杂志;2011年08期
本文编号：2275066
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