本文選題：流感　切入點(diǎn)：亞單位疫苗　出處：《中國(guó)疾病預(yù)防控制中心》2010年博士論文　論文類型：學(xué)位論文

【摘要】： 廣譜流感疫苗對(duì)防控流感大流行具有重要意義。十幾年來,廣譜流感疫苗的研究取得了重要進(jìn)展。本課題研發(fā)的在大腸桿菌中表達(dá)的NP與M2e融合蛋白NM2e的亞單位疫苗,因生產(chǎn)成本低、可大規(guī)模生產(chǎn)和反復(fù)使用等優(yōu)點(diǎn),具有良好的開發(fā)前景。但這種疫苗免疫原性弱,需要佐劑才能誘發(fā)有效和長(zhǎng)期的免疫保護(hù)。 鋁佐劑是目前最廣泛應(yīng)用的一類疫苗佐劑,但主要誘發(fā)Th2型免疫反應(yīng)。CpG是被廣泛研究過的Th1型佐劑,能誘發(fā)較強(qiáng)的細(xì)胞免疫,但目前尚未被批準(zhǔn)市售。一些化學(xué)藥物如左旋咪唑(Levamisole, LMS)、西米替丁(Cimetidine, Cim)、伐地那非(Vardenafil, Var,品牌名：艾力達(dá))、PolyⅠ：C等在人體中具有一定的免疫調(diào)節(jié)作用,文獻(xiàn)報(bào)道這類化學(xué)藥物對(duì)乙肝抗原具有明顯的佐劑效應(yīng)。由于這類藥物價(jià)格低廉,有人體長(zhǎng)期應(yīng)用經(jīng)驗(yàn),毒副作用清楚,一旦開發(fā)成為佐劑,將具有重要應(yīng)用前景。因此,對(duì)這類藥物在蛋白亞單位疫苗中佐劑效應(yīng)的研究已成為佐劑研究的新策略。為此,本研究選用了NM2e為抗原,對(duì)Al(OH)3、CpG、LMS、Cim、Var、Poly I:C的佐劑效應(yīng)進(jìn)行比較研究。 本研究首先對(duì)NM2e蛋白進(jìn)行了批量純化,使用純化抗原對(duì)Al(OH)3與CpG的佐劑效應(yīng)進(jìn)行了研究,在此基礎(chǔ)上將LMS、Cim、Var、Poly I:C四種藥物單獨(dú)或與Al(OH)3聯(lián)合制備NM2e亞單位疫苗,通過免疫小鼠的體液免疫、細(xì)胞免疫及攻毒后的保護(hù)效果,對(duì)這四種藥物作為NM2e蛋白疫苗佐劑的可能性進(jìn)行了比較研究。主要研究結(jié)果如下： 1、利用表達(dá)條件IPTG=0.1mM, OD600=0.8, t=10h, T=25℃能使原核表達(dá)質(zhì)粒pET30a-NM2e可溶性表達(dá)NM2e蛋白,用陰離子交換層析和凝膠過濾層析兩步純化工藝,從10L菌液中批量制備獲得了質(zhì)量一致、純度為90%的70mg NM2e蛋白,經(jīng)Western-blot鑒定蛋白正確。 2. Al(OH)3佐劑可以顯著提高NM2e蛋白誘發(fā)的特異性IgGl滴度,可使攻毒后小鼠的存活率提高至70%(7／10)。CpG佐劑可以明顯提高NM2e蛋白誘發(fā)的特異性IgG2a滴度,攻毒后小鼠的存活率提高至30%(3/10)。Al(OH)3與CpG聯(lián)合使用作為佐劑,NM2e蛋白的特異性IgG1、IgG2a滴度顯著高于CpG佐劑組,NM2e蛋白的特異性IgGl與單獨(dú)使用Al(OH)3無顯著差別,但I(xiàn)gG2a滴度高于Al(OH)3佐劑組,攻毒后小鼠發(fā)病最輕,體重恢復(fù)最快,小鼠的存活率為100%。 3、左旋咪唑?qū)M2e蛋白亞單位疫苗無明顯佐劑效應(yīng),但與Al聯(lián)合使用可使攻毒后存活小鼠體重恢復(fù)加快。西米替丁可以明顯提高NM2e蛋白誘發(fā)的特異性IgG2a滴度,提高攻毒后小鼠的存活率(30%),但保護(hù)效果明顯低于Al(70%),與Al無明顯協(xié)同作用。伐地那非能明顯增加NM2e蛋白誘發(fā)的特異性IgG1、IgG2a滴度,提高攻毒后小鼠的存活率(36%),但與Al聯(lián)合使用降低了Al的免疫保護(hù)。Poly I:C能明顯增加NM2e蛋白誘發(fā)的特異性IgG滴度,傾向于提高Th1型免疫反應(yīng),能提高攻毒后小鼠的存活率(20%),但保護(hù)效果明顯低于Al,與Al聯(lián)合使用對(duì)小鼠的攻毒保護(hù)率有一定程度的提高。 以上實(shí)驗(yàn)結(jié)果證明,NM2e蛋白在大腸桿菌中獲得了批量表達(dá)和純化；Al和CpG佐劑均可以增強(qiáng)NM2e蛋白的免疫反應(yīng),均能提高攻毒保護(hù)效果,二者聯(lián)合使用具有明顯的協(xié)同作用；左旋咪唑、西米替丁、伐地那非、Poly I:C對(duì)NM2e蛋白均有一定的免疫調(diào)節(jié)作用,其中西米替丁和伐地那非能提高攻毒保護(hù)效果,但保護(hù)效果明顯均低于Al,與Al聯(lián)合使用均無明顯協(xié)同作用,Poly I:C與Al聯(lián)合使用的的協(xié)同作用尚需進(jìn)一步研究。
[Abstract]:Influenza vaccine is important for the prevention and control of influenza pandemic. Over the past decade, important progress has been made in the research of broad-spectrum influenza vaccine. The subunit NP and M2e expression in Escherichia coli and NM2e fusion protein vaccine, due to low production cost, large-scale production and repeated use of the advantages of good development future. But this vaccine weak immunogenicity, need adjuvant to induce effective and long-term immune protection.
Aluminum is a kind of adjuvant vaccine adjuvants is currently the most widely used, but the main inducing immune response to Th2.CpG is Th1 type adjuvant widely studied, it can induce strong cellular immunity, but has not yet been approved commercially. Some chemical drugs such as levamisole (Levamisole, LMS), cimetidine (Cimetidine Cim), vardenafil (Vardenafil, Var, brand name: Poly 1: C, Levitra) in the human body has a certain role in immune regulation, reports of these chemical substances has obvious effect on adjuvant hepatitis B antigen. This kind of drugs because the price is low, a long-term application of human experience, side the role of clear, once developed as adjuvants, will have important application prospects. Therefore, to study this kind of drug in the adjuvant effect of the subunit vaccine has become a new strategy for adjuvant research. Therefore, this study selected NM2e as antigen of Al (OH) 3, CpG, LMS The adjuvant effects of Cim, Var, and Poly I:C were compared.
This study first conducted batch purification of NM2e protein using purified antigen of Al (OH) 3 and the adjuvant effects of CpG were studied. On the basis of LMS, Cim, Var, Poly, I:C four kinds of drugs alone or with Al (OH) 3 combined preparation of NM2e subunit vaccine, humoral immunity by immunization of mice the protective effect of cell immunity and infection after the comparative study of these four drugs as potential NM2e vaccine adjuvant was carried out. The main results are as follows:
1, the expression of OD600=0.8, t=10h, IPTG=0.1mM, T=25 C can make the prokaryotic plasmid pET30a-NM2e soluble protein expression of NM2e, chromatography and gel filtration chromatography two step purification by anion exchange, from 10L solution batch preparation obtained the same quality, purity of 90% 70mg NM2e protein identified by Western-blot protein.
2. Al (OH) 3 adjuvants can significantly improve the specificity of IgGl was induced by NM2e protein, the survival rate of mice after infection increased to 70% (7 / 10).CpG adjuvant can significantly improve the specificity of the titer of IgG2a NM2e protein induced after infection, mice survival rate increased to 30% (3/10).Al (OH) the combination of 3 and CpG as adjuvant, the specificity of IgG1 NM2e protein. The titer of IgG2a was significantly higher than that of CpG adjuvant group, the specificity of IgGl NM2e protein and Al alone (OH) 3 had no significant difference, but the titer of IgG2a was higher than that of Al (OH) 3 adjuvant group challenged mice after the onset of light. Weight recovery is the fastest, the survival rate of mice was 100%.
3, levamisole had no obvious effect on adjuvant NM2e protein subunit vaccine, but combined with the Al can make the weight recovery of virus challenged mice accelerated. Cimetidine can significantly improve the specificity of IgG2a titer of NM2e protein induced, improve the survival rate of mice after infection (30%), but the protective effect was significantly lower than that of Al (70%), no obvious synergistic effect with Al. Vardenafil could significantly increase the specificity of IgG1, NM2e protein induced by IgG2a titer, improve the survival rate of mice after infection (36%), but with the combination of Al reduced the specific titer of IgG.Poly I:C Al immune protection can significantly increase the NM2e protein induced, tend to enhance the immune response to Th1, can improve the survival rate of mice after infection (20%), but the protective effect was significantly lower than that of Al, and the combined use of Al attack protection on mice was improved to a certain extent.
The experiment results proved that the NM2e protein obtained batch expression in Escherichia coli and purified; Al and CpG adjuvant can enhance immune response of NM2e protein, can enhance the protective effect, the combined use of the two have obvious synergistic effect; levamisole, cimetidine, vardenafil, Poly I:C the immunomodulatory effects of NM2e protein were found, which cimetidine and vardenafil can improve the protective effect, but the protective effects were obviously lower than that of Al, and Al had no obvious synergistic effect of combined use of Poly, I:C and Al combined with the synergy needed further study.

【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】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 付麗麗;盧錦標(biāo);楊蕾;陳保文;都偉欣;鄧海清;王國(guó)治;黃長(zhǎng)江;;氫氧化鋁和PolyIC佐劑在結(jié)核亞單位疫苗中的佐劑效應(yīng)[J];中國(guó)生物制品學(xué)雜志;2014年03期

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本文編號(hào)：1588577