噬菌體展示候選甲型流感病毒和乙型流感病毒聯(lián)合疫苗的構(gòu)建及免疫原性研究
發(fā)布時(shí)間：2018-10-16 17:08
【摘要】： 流行性感冒是一種非常嚴(yán)重的全球性的呼吸道傳染病。在流感流行期間，全球有10—20％的人口感染流感病毒，每年造成三百萬至五百萬嚴(yán)重病例和250，000至500，000例死亡。對(duì)流感的預(yù)防尚無非常有效的方法，接種疫苗是目前最有效的途徑。血凝素(HA)和神經(jīng)氨酸(NA)是存在于病毒表面易感的大型糖蛋白，且具有強(qiáng)的免疫原性，是非常好的疫苗抗原。現(xiàn)在市場(chǎng)上的疫苗都將血凝素(hemagglutinin，HA)和神經(jīng)氨酸(neuraminidase，NA)作為主要或唯一病毒抗原。血凝素(HA)和神經(jīng)氨酸(NA)基因的改變和重排，是通過疫苗接種控制流感流行的主要障礙。因此，流感病毒能夠重復(fù)感染并使得每年需要新的疫苗進(jìn)行免疫接種。開發(fā)基于流感病毒不變區(qū)域，能產(chǎn)生廣譜的交叉免疫反應(yīng)的普適性流感疫苗是迫切需要的。 由病毒基因編碼的M2蛋白是一個(gè)四聚體離子通道，在病毒顆粒中少量表達(dá)或不表達(dá)，但卻在被病毒感染的細(xì)胞表面大量表達(dá)。M2蛋白膜外區(qū)(M2e)由24個(gè)氨基酸殘基組成，并且不發(fā)生基因漂移和變異。自1918年西班牙流感大流行至今，雖然其間發(fā)生了無數(shù)次的流感流行和三次全球性大流行，M2e序列基本保持不變。研究表明，不同構(gòu)造形式的基于M2e的疫苗能夠提供保護(hù)性免疫。乙型流感病毒的BM2蛋白由109個(gè)氨基酸殘基組成，是乙型流感病毒編碼的血凝素(HA)、神經(jīng)氨酸(NA)和NB糖蛋白之外的第四個(gè)膜內(nèi)在蛋白。BM2具有離子通道活性，熒光顯微鏡顯示BM2表達(dá)于為感染細(xì)胞表面。雖然BM2在乙型流感病毒生活周期中有著自己特有的功能，但是BM2還是被認(rèn)為具有和M2蛋白在甲型流感病毒中相似的功能，可能能作為通用乙型流感病毒的候選表位。 流感病毒研究中常用的是小鼠動(dòng)物模型不能在小鼠間傳播，而豚鼠不但能對(duì)未經(jīng)適應(yīng)性改變的流感病毒敏感，流感病毒也能在豚鼠中以飛沫傳播，因此豚鼠可以作為流感病毒傳染研究的新動(dòng)物模型。噬菌體疫苗是以噬菌體作為疫苗載體，將特異表位展示在噬菌體顆粒表面，構(gòu)建成不同表位特異的噬菌體疫苗，具有病毒樣顆粒特性，能引發(fā)強(qiáng)的針對(duì)表位的特異性免疫應(yīng)答。鼻腔免疫是近年發(fā)展起來新的粘膜免疫途徑，它通過抗原在鼻粘膜的釋放，誘導(dǎo)系統(tǒng)和粘膜免疫應(yīng)答。研究表明，由T細(xì)胞、B細(xì)胞、樹突細(xì)胞以及包括M細(xì)胞的隱窩吸收上皮細(xì)胞組成的鼻粘膜相關(guān)淋巴組織(nose associated lymphoid tissue，NALT)，是鼻腔免疫的誘導(dǎo)部位。殼聚糖是一個(gè)新型的緩控釋疫苗呈遞系統(tǒng)載體，能安全而有效地促進(jìn)鼻腔吸收微球疫苗。 本文利用基因重組將M2eBM2多表位基因片斷與T7 10B衣殼蛋白基因融合構(gòu)建噬菌體候選疫苗，PCR鑒定為陽性的噬菌體候選疫苗擴(kuò)增后用甲醛滅活。用10~(10)pfu劑量的噬菌體微球聯(lián)合流感候選疫苗經(jīng)鼻腔免疫豚鼠，并用10~3pfu的H3N2、H1N1和B型流感病毒分別對(duì)經(jīng)候選疫苗免疫和未經(jīng)候選疫苗免疫的豚鼠進(jìn)行攻擊，分別檢測(cè)上呼吸道(upstream respiratory tract，URT)和肺部流感病毒滴度，，對(duì)比研究候選疫苗對(duì)病毒感染的保護(hù)作用。 ELISA檢測(cè)血清IgG水平發(fā)現(xiàn)，噬菌體微球聯(lián)合流感候選疫苗具有特異性免疫原性，能誘導(dǎo)產(chǎn)生針對(duì)M2e和BM2的特異性抗體。對(duì)病毒感染的豚鼠呼吸道內(nèi)的病毒滴度檢測(cè)表明，噬菌體微球聯(lián)合流感候選疫苗對(duì)豚鼠的全呼吸道具有抗H3N2和H1N1的保護(hù)作用；對(duì)乙型流感病毒感染的豚鼠上呼吸道候選疫苗無保護(hù)效果，肺部有一定的保護(hù)效果。 因此，(1)構(gòu)建的噬菌體微球聯(lián)合流感候選疫苗經(jīng)鼻腔免疫能有效誘導(dǎo)特異性體液免疫，產(chǎn)生特異性抗體(M2eBM2)；(2)候選疫苗誘導(dǎo)的M2e抗體對(duì)感染H3N2和H1N1的豚鼠的全呼吸道有保護(hù)作用；(3)候選疫苗誘導(dǎo)的BM2抗體對(duì)感染乙流感病毒的豚鼠肺部有保護(hù)作用而對(duì)上呼吸道無保護(hù)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
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392
本文編號(hào)：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
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392
【引證文獻(xiàn)】
相關(guān)期刊論文 前2條
1 湯洋;王海漩;烏美妮;胡凝珠;胡云章;;流感病毒感染豚鼠模型的初步建立[J];中國生物制品學(xué)雜志;2011年03期
2 王海漩;烏美妮;湯洋;李建芳;李彥涵;胡云章;趙蕊蕊;沈霏;段志青;胡凝珠;;廣譜流感噬菌體微球疫苗對(duì)豚鼠的免疫保護(hù)作用[J];中國生物制品學(xué)雜志;2011年08期
本文編號(hào)：2275066
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