提高广谱流感疫苗NM2e免疫效果的佐剂研究

发布时间：2018-03-09 12:35

  本文选题：流感　切入点：亚单位疫苗　出处：《中国疾病预防控制中心》2010年博士论文　论文类型：学位论文

【摘要】： 广谱流感疫苗对防控流感大流行具有重要意义。十几年来,广谱流感疫苗的研究取得了重要进展。本课题研发的在大肠杆菌中表达的NP与M2e融合蛋白NM2e的亚单位疫苗,因生产成本低、可大规模生产和反复使用等优点,具有良好的开发前景。但这种疫苗免疫原性弱,需要佐剂才能诱发有效和长期的免疫保护。 铝佐剂是目前最广泛应用的一类疫苗佐剂,但主要诱发Th2型免疫反应。CpG是被广泛研究过的Th1型佐剂,能诱发较强的细胞免疫,但目前尚未被批准市售。一些化学药物如左旋咪唑(Levamisole, LMS)、西米替丁(Cimetidine, Cim)、伐地那非(Vardenafil, Var,品牌名：艾力达)、PolyⅠ：C等在人体中具有一定的免疫调节作用,文献报道这类化学药物对乙肝抗原具有明显的佐剂效应。由于这类药物价格低廉,有人体长期应用经验,毒副作用清楚,一旦开发成为佐剂,将具有重要应用前景。因此,对这类药物在蛋白亚单位疫苗中佐剂效应的研究已成为佐剂研究的新策略。为此,本研究选用了NM2e为抗原,对Al(OH)3、CpG、LMS、Cim、Var、Poly I:C的佐剂效应进行比较研究。 本研究首先对NM2e蛋白进行了批量纯化,使用纯化抗原对Al(OH)3与CpG的佐剂效应进行了研究,在此基础上将LMS、Cim、Var、Poly I:C四种药物单独或与Al(OH)3联合制备NM2e亚单位疫苗,通过免疫小鼠的体液免疫、细胞免疫及攻毒后的保护效果,对这四种药物作为NM2e蛋白疫苗佐剂的可能性进行了比较研究。主要研究结果如下： 1、利用表达条件IPTG=0.1mM, OD600=0.8, t=10h, T=25℃能使原核表达质粒pET30a-NM2e可溶性表达NM2e蛋白,用阴离子交换层析和凝胶过滤层析两步纯化工艺,从10L菌液中批量制备获得了质量一致、纯度为90%的70mg NM2e蛋白,经Western-blot鉴定蛋白正确。 2. Al(OH)3佐剂可以显著提高NM2e蛋白诱发的特异性IgGl滴度,可使攻毒后小鼠的存活率提高至70%(7／10)。CpG佐剂可以明显提高NM2e蛋白诱发的特异性IgG2a滴度,攻毒后小鼠的存活率提高至30%(3/10)。Al(OH)3与CpG联合使用作为佐剂,NM2e蛋白的特异性IgG1、IgG2a滴度显著高于CpG佐剂组,NM2e蛋白的特异性IgGl与单独使用Al(OH)3无显著差别,但IgG2a滴度高于Al(OH)3佐剂组,攻毒后小鼠发病最轻,体重恢复最快,小鼠的存活率为100%。 3、左旋咪唑对NM2e蛋白亚单位疫苗无明显佐剂效应,但与Al联合使用可使攻毒后存活小鼠体重恢复加快。西米替丁可以明显提高NM2e蛋白诱发的特异性IgG2a滴度,提高攻毒后小鼠的存活率(30%),但保护效果明显低于Al(70%),与Al无明显协同作用。伐地那非能明显增加NM2e蛋白诱发的特异性IgG1、IgG2a滴度,提高攻毒后小鼠的存活率(36%),但与Al联合使用降低了Al的免疫保护。Poly I:C能明显增加NM2e蛋白诱发的特异性IgG滴度,倾向于提高Th1型免疫反应,能提高攻毒后小鼠的存活率(20%),但保护效果明显低于Al,与Al联合使用对小鼠的攻毒保护率有一定程度的提高。 以上实验结果证明,NM2e蛋白在大肠杆菌中获得了批量表达和纯化；Al和CpG佐剂均可以增强NM2e蛋白的免疫反应,均能提高攻毒保护效果,二者联合使用具有明显的协同作用；左旋咪唑、西米替丁、伐地那非、Poly I:C对NM2e蛋白均有一定的免疫调节作用,其中西米替丁和伐地那非能提高攻毒保护效果,但保护效果明显均低于Al,与Al联合使用均无明显协同作用,Poly I:C与Al联合使用的的协同作用尚需进一步研究。
[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.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

【引证文献】

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1 付丽丽;卢锦标;杨蕾;陈保文;都伟欣;邓海清;王国治;黄长江;;氢氧化铝和PolyIC佐剂在结核亚单位疫苗中的佐剂效应[J];中国生物制品学杂志;2014年03期

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