本文選題：H5N1 + 桿狀病毒載體疫苗�。� 參考：《中國(guó)疾病預(yù)防控制中心》2012年碩士論文

【摘要】：自1997年首次報(bào)道高致病性禽流感H5N1病毒能夠直接感染人并導(dǎo)致死亡事件以來(lái),在過(guò)去的十多年內(nèi),H5N1在全球范圍內(nèi)傳播的不可預(yù)測(cè)性、對(duì)家禽和人類的高致病性以及病毒復(fù)雜的進(jìn)化和重配等特點(diǎn),充分說(shuō)明了它可導(dǎo)致流感大流行的可能。毫無(wú)疑問(wèn),疫苗接種能夠?yàn)闈撛诘腍5N1流感大流行的爆發(fā)提供最有效的防控手段。而基于雞胚生產(chǎn)的傳統(tǒng)型流感疫苗由于生產(chǎn)能力有限等因素,無(wú)法滿足流感大流行發(fā)生后的快速需求。因此,亟需開(kāi)發(fā)出一種新型流感疫苗生產(chǎn)體系來(lái)及時(shí)生產(chǎn)流感大流行疫苗。 為了獲取免疫原性更強(qiáng)、保護(hù)效果更廣以及生產(chǎn)系統(tǒng)更為快速的禽流感疫苗,本研究基于桿狀病毒表面展示技術(shù),開(kāi)展了針對(duì)禽流感新型疫苗的初步研究。主要研究?jī)?nèi)容如下： (1)利用改造的pFast BacTM Dual載體質(zhì)粒構(gòu)建pFast-HA-NA表達(dá)質(zhì)粒,通過(guò)Bac-to-Bac桿狀病毒表達(dá)系統(tǒng),在其表面共同展示禽流感病毒H5N1亞型(A/Hubei/1/2010)的主要免疫原性蛋白HA和NA,制備出新型禽流感疫苗Bac-HA-NA; (2)采用BALB/c小鼠模型評(píng)價(jià)該疫苗的免疫原性。通過(guò)兩種策略進(jìn)行免疫,即肌肉注射100μ1Bac-HA-NA (HA血凝滴度Log28)和滴鼻免疫25μ1Bac-HA-NA (HA血凝滴度Log210),二周后對(duì)每組小鼠進(jìn)行加強(qiáng)免疫,免疫方式、免疫劑量與第一次免疫完全相同。利用間接ELISA實(shí)驗(yàn)檢測(cè)血清中HA特異性IgG抗體滴度,ELISPOT實(shí)驗(yàn)檢測(cè)小鼠淋巴細(xì)胞分泌IFN-y能力。 (3)利用A/PR/8/34H1N1病毒對(duì)小鼠進(jìn)行攻擊實(shí)驗(yàn),評(píng)價(jià)該疫苗的交叉保護(hù)效率。 研究結(jié)果表明： 1)載體疫苗Bac-HA-NA的桿狀病毒表面能夠成功的同時(shí)表達(dá)HA和NA蛋白,HA蛋白大小為72kDa, NA蛋白大小為55kDa,并且表達(dá)的HA蛋白血凝滴度達(dá)到log211,表達(dá)的NA蛋白與重配的湖北株H5N1有相似的NA酶活特性,對(duì)奧司他韋和扎那米韋有相似的藥物敏感性。 2) Bac-HA-NA載體疫苗采用肌肉注射途徑在小鼠體內(nèi)誘導(dǎo)的HA特異性IgG抗體滴度達(dá)到1：40000,而滴鼻免疫途徑在小鼠體內(nèi)的HA特異性IgG抗體水平為陰性結(jié)果。 3)肌肉注射途徑能夠在小鼠體內(nèi)誘導(dǎo)較強(qiáng)的細(xì)胞免疫水平,達(dá)到479SFCs/million splenocytes。滴鼻免疫途徑也能夠在小鼠體內(nèi)產(chǎn)生一定的細(xì)胞免疫水平,達(dá)到325SFCs/million splenocytes。 4)肌注免疫對(duì)A/PR/8/34(H1N1)的交叉保護(hù)效果能達(dá)到25%,而滴鼻免疫則無(wú)交叉保護(hù)效果。 本研究結(jié)果表明,基于桿狀病毒表面展示系統(tǒng)構(gòu)建的流感載體疫苗Bac-HA-NA,可以產(chǎn)生有效的體液免疫和細(xì)胞免疫,并具有部分交叉保護(hù)作用。由于桿狀病毒生產(chǎn)系統(tǒng)的高安全性、易于分離純化,生產(chǎn)周期短而顯優(yōu)于傳統(tǒng)流感疫苗生產(chǎn)系統(tǒng),可以用于將來(lái)的流感大流行疫苗的生產(chǎn)。
[Abstract]:Since 1997, when the highly pathogenic avian influenza H5N1 virus was first reported to be capable of directly infecting humans and causing deaths, the global spread of H5N1 has been unpredictable over the past decade or so.The high pathogenicity of poultry and humans and the complex evolution and reassortment of viruses fully illustrate the possibility that it can lead to influenza pandemic.There is no doubt that vaccination can provide the most effective means of prevention and control for a potential H5N1 pandemic.The traditional influenza vaccine based on chicken embryo can not meet the rapid demand of influenza pandemic due to limited production capacity and other factors.Therefore, it is urgent to develop a new influenza vaccine production system to produce influenza pandemic vaccine in time.In order to obtain avian influenza vaccine with stronger immunogenicity, wider protective effect and faster production system, a preliminary study on new avian influenza vaccine was carried out based on baculovirus surface display technology.The main contents of the study are as follows:1) the pFast-HA-NA expression plasmid was constructed by using the modified pFast BacTM Dual vector plasmid. By Bac-to-Bac baculovirus expression system, the main immunogenicity proteins HA and NAA of H5N1 subtype A / Hubei / 1 / 2010) were jointly displayed on its surface, and a new avian influenza vaccine, HBac-HA-NAA, was prepared.BALB/c mouse model was used to evaluate the immunogenicity of the vaccine.Two strategies were used to immunize the mice: intramuscular injection of 100 渭 1Bac-HA-NA HA hemagglutination titer Log28) and nasal immunization with 25 渭 1Bac-HA-NA HA hemagglutination titer Log210. After two weeks, the mice in each group were immunized more intensively, and the immunization mode was exactly the same as that in the first immunization.Indirect ELISA assay was used to detect the titer of HA specific IgG antibody in serum. Elispot assay was used to detect the ability of murine lymphocytes to secrete IFN-y.A/PR/8/34H1N1 virus was used to attack mice to evaluate the cross protection efficiency of the vaccine.The results show that:1) the baculovirus surface of the vector vaccine Bac-HA-NA can successfully express HA and na protein at the same time. The size of HA protein is 72 kDa. na protein size is 55 kDa. the HA protein hemagglutination titer of expressed HA protein reaches log211, and the expressed na protein is matched with H5N1 of Hubei strain.Have similar characteristics of na enzyme activity,There is a similar sensitivity to oseltamivir and zanamivir.2) the titer of HA specific IgG antibody induced by Bac-HA-NA vector vaccine in mice by intramuscular injection was 1: 40000, but the level of HA-specific IgG antibody induced by nasal drip was negative in mice.3) the intramuscular injection pathway could induce strong cellular immunity in mice and reach 479SFCs/million splenocytes.Nasal drip immune pathway can also produce a certain level of cellular immunity in mice, reaching 325SFCs/million splenocytes.4) the cross protective effect of intramuscular immunization on Ar / R / 8 / 34 H1 was 25%, but nasal drip had no cross protective effect.The results show that the influenza vector vaccine Bac-HA-NAbased on baculovirus surface display system can produce effective humoral and cellular immunity and has partial cross-protection.Because of the high safety of baculovirus production system, easy separation and purification, the production cycle is short and superior to the traditional influenza vaccine production system, so it can be used in the production of influenza pandemic vaccine in the future.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉永平;王方海;蘇志堅(jiān);李廣宏;龐義;;昆蟲桿狀病毒表達(dá)載體系統(tǒng)的研究及應(yīng)用[J];昆蟲知識(shí);2006年01期

，

本文編號(hào)：1759460