【摘要】： 流感是由流感病毒引起的急性呼吸道傳染病。幾十年來,人流感的流行一直以A型H3/H1亞型以及B型流感為主,禽流感的流行以A型H5/H7/H9亞型為主,其流行已造成了巨大的損失。近年來,禽流感病毒的多個(gè)亞型(H5/H7/H9/H10)開始不斷突破種間屏障感染人類,并可能造成大流行。當(dāng)前,傳統(tǒng)的流感疫苗已無法有效應(yīng)對(duì)這一趨勢(shì),各國(guó)學(xué)者紛紛進(jìn)行新型流感疫苗的開發(fā)以應(yīng)對(duì)流感跨種傳播及多亞型并存的現(xiàn)狀。 本論文即擬對(duì)多亞型流感病毒復(fù)合多表位核酸疫苗進(jìn)行研究。在搜集已發(fā)表流感功能表位的基礎(chǔ)上,應(yīng)用生物信息學(xué)理論對(duì)H1/H3/H5/H7/H9亞型的保護(hù)性抗原血凝素及神經(jīng)氨酸酶進(jìn)行了CTL、Th、B細(xì)胞表位的預(yù)測(cè)和篩選分析。設(shè)計(jì)組建了多表位功能基因盒,并進(jìn)行了人工模擬優(yōu)化,應(yīng)用體外實(shí)驗(yàn)方式分析了預(yù)測(cè)的Th和B細(xì)胞功能表位抗原性。根據(jù)針對(duì)所屬動(dòng)物及預(yù)防目的的不同,我們構(gòu)建了三種主導(dǎo)型H3/H1(針對(duì)人),H5/H3(針對(duì)人和豬),H5/H7(針對(duì)人和禽)的復(fù)合多表位核酸疫苗,通過RT-PCR及間接免疫熒光方法證實(shí)疫苗中各抗原組分可有效表達(dá)。最后,我們將所構(gòu)建的復(fù)合多表位核酸疫苗進(jìn)行了小鼠、豬、雞的實(shí)驗(yàn)免疫研究,通過對(duì)ELISA及HI抗體水平、淋巴細(xì)胞刺激指數(shù)、IFNγ-ELISPOT和T淋巴細(xì)胞亞類數(shù)量、血清細(xì)胞因子含量等指標(biāo)的綜合分析,對(duì)不同復(fù)合多表位疫苗進(jìn)行了免疫原性評(píng)價(jià)。結(jié)果顯示,復(fù)合多表位疫苗組在針對(duì)主要抗原的抗體水平與不含表位組相當(dāng)?shù)那闆r下,表位相關(guān)抗原的抗體水平顯示了一定的優(yōu)勢(shì)。表位相關(guān)抗原的淋巴細(xì)胞刺激指數(shù)顯著升高,IFNγ-ELISPOT斑點(diǎn)數(shù)不同程度增加。復(fù)合多表位疫苗免疫組CD4~+T淋巴細(xì)胞數(shù)量和血清Th2類細(xì)胞因子含量顯著增加。以上結(jié)果證實(shí),復(fù)合多表位疫苗在誘導(dǎo)細(xì)胞及體液免疫方面均顯示了優(yōu)勢(shì),與各免疫對(duì)照組相比復(fù)合多表位DNA疫苗誘導(dǎo)的機(jī)體整體免疫水平最優(yōu)。本研究對(duì)可同時(shí)預(yù)防多種亞型流感病毒,可對(duì)多種動(dòng)物免疫保護(hù),可提供機(jī)體長(zhǎng)久免疫保護(hù)的跨種通用流感疫苗進(jìn)行了有益嘗試。
[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 緯-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 緯-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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 譚磊;魯會(huì)軍;張丹;王開艷;田明堯;劉存霞;劉燕瑜;胡博;金寧一;;季節(jié)性和大流行性H1N1流感血凝素DNA疫苗電穿孔免疫小鼠及攻毒保護(hù)實(shí)驗(yàn)[J];中國(guó)科學(xué):生命科學(xué);2011年02期

相關(guān)博士學(xué)位論文 前1條

1 譚磊;流感H3/H1亞型多表位核酸疫苗和重組腺病毒疫苗構(gòu)建及實(shí)驗(yàn)免疫研究[D];吉林大學(xué);2011年

相關(guān)碩士學(xué)位論文 前1條

1 王開艷;H3/H1亞型流感核酸疫苗的構(gòu)建及小鼠免疫試驗(yàn)[D];延邊大學(xué);2010年

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本文編號(hào)：2350940