新型免疫佐剂的研制及亚单位佐剂疫苗的免疫效力检验

发布时间：2018-03-10 00:32

本文选题：佐剂　切入点：免疫增强　出处：《中国海洋大学》2012年硕士论文　论文类型：学位论文

【摘要】：疫苗免疫是现今预防动物传染病的最有效的手段之一。动物疫苗的生产研究具有重大的社会经济效益。佐剂作为一种免疫增强剂，成为绝大部分疫苗不可或缺的一部分。新型佐剂也随着疫苗市场的拓展，具有广阔的前景。国内佐剂研究不断深入，有研究验证了一些常规佐剂在亚单位疫苗中的应用效果，但自主研发的商品化佐剂较少，很多动物疫苗的生产依赖进口佐剂。传统的乳化方法，增加了企业能耗，使得成本过高。本研究就是基于生产所需的高安全性、高免疫增强性、低能耗、低成本的要求，设计研发了一组油乳剂佐剂和一款高通量制备的富含猪特异性CpG的佐剂，以期开发适合猪口蹄疫亚单位疫苗的商品化佐剂。本研究主要采用了HLB法对复合乳剂形成的各组配方进行筛选，确定最佳方案；在制备微乳剂佐剂时主要通过三元相图法绘制微乳区域来寻求最优配伍。复合油佐剂COA，为一款由更安全的油相成分和毒性较小的乳化剂混合而成的油佐剂，其乳化工艺及乳剂性质类似法国Seppic生产的ISA206佐剂，低速搅拌即可乳化；W/O/W剂型使乳剂即时、持续释放抗原，用以迅速持久的免疫。 MOM佐剂，为一款矿物油微乳佐剂，为自乳化体系。油水相混合均匀，即可形成纳米级微乳，稳定性极高。W/O剂型和极小颗粒有利于抗原的缓慢释放，提高免疫。 MFM佐剂，为一款用可代谢动物油配制的微乳佐剂。作为MF59佐剂的改进配方，它同样是自乳化体系，油水相混匀即可乳化，乳剂颗粒达到纳米级，稳定性极高。以上3款油佐剂根据商品化佐剂即开即用的需求设计，乳化简单，稳定性强。 CpG-sw佐剂，为一款通过基因工程手段，设计制备的富含猪特异性CpG基序的核酸佐剂，同时借助工程菌增殖提高CpG生产通量，提升了CpG的可利用度。本研究将试制的几款佐剂合并胸腺素，形成制剂类、核酸类、蛋白类三类典型的佐剂系统，以传统白油佐剂作为阳性对照，无佐剂抗原作为阴性对照分别与亚单位疫苗制成佐剂疫苗，免疫小鼠，经体内实验，以初步筛选出合适的亚单位疫苗佐剂。经过动物实验验证，发现MOM组、白油阳性组和CpG-sw组小鼠在免疫初期出现全身反应，1~2日内反应消失。MOM组和白油阳性组在注射部位产生硬结，难以消化，MOM硬结较小。其他组未见反应。制剂外相的亲水性和佐剂本身的毒性影响到佐剂的安全性。从小鼠IgG抗体水平来看，MOM组、白油阳性组和CpG-sw组抗体水平最高，其他各组较阴性组也有明显提高，发挥了佐剂作用。从脾细胞细胞因子浓度来看，CpG组、Tα1组细胞因子浓度较阴性轻微提高，但提高不明显，各油佐剂组均有下降，但下降不明显。不同佐剂在刺激T、B淋巴细胞反应方面也表现不一。这几款典型佐剂特点各异，在剂型、水溶性、免疫机制方面都有不同，体现出不同免疫增强的途径、趋势和特点。
[Abstract]:Vaccine immunization is one of the most effective methods to prevent animal infectious diseases. Research on animal vaccine production has great social and economic benefits. As an indispensable part of most vaccines, new adjuvants have broad prospects with the development of vaccine market. The domestic research on adjuvant has been deepening. Some studies have verified the application effect of some conventional adjuvants in subunit vaccines, but the commercial adjuvants developed by ourselves are relatively few. Many animal vaccines are produced by imported adjuvants. Traditional emulsification methods. Increases the enterprise energy consumption, causes the cost to be too high. Based on the requirements of high safety, high immunity enhancement, low energy consumption and low cost for production, a group of oil emulsion adjuvants and a high-throughput adjuvant rich in porcine specific CpG were designed and developed. To develop a commercial adjuvant for porcine foot-and-mouth disease subunit vaccine. In this study, the HLB method was used to screen the formulations of the composite emulsion and to determine the best formula, and the ternary phase diagram method was used to draw the region of the microemulsion to find the best compatibility in the preparation of the microemulsion adjuvant. The compound oil adjuvant, COA, is an oil adjuvant made from a safer oil phase and a less toxic emulsifier. Its emulsifying process and emulsion properties are similar to those of the ISA206 adjuvant produced by Seppic in France. The emulsion can be emulsified by low speed stirring in the form of W / O / W / W. The persistent release of antigens is used for rapid and persistent immunity. MOM adjuvant is a kind of mineral oil microemulsion adjuvant, which is self-emulsifying system. When the oil and water are mixed evenly, the nanometer microemulsion can be formed. MFM adjuvant is a microemulsion adjuvant prepared from metabolizable animal oil. As an improved formula of MF59 adjuvant, it is also a self-emulsifying system. The above three oil adjuvants are designed according to the requirements of commercial adjuvants, easy emulsification and strong stability. CpG-sw adjuvant is a kind of nucleic acid adjuvant which is rich in porcine specific CpG motifs designed and prepared by genetic engineering method. At the same time, the production flux of CpG is increased with the help of the multiplication of engineering bacteria, and the availability of CpG is improved. In this study, several adjuvants were combined with thymosin to form three typical adjuvant systems: preparation, nucleic acid and protein. The traditional white oil adjuvant was used as positive control. Adjuvant free antigen was used as negative control to make adjuvant vaccine with subunit vaccine respectively. The mice were immunized with adjuvant antigen. The suitable adjuvant of subunit vaccine was screened in vivo. After animal experiment, it was found that the systemic reaction disappeared within 1 ~ 2nd in MOM group, white oil positive group and CpG-sw group. The other groups have no reaction. The hydrophilicity of the preparation and the toxicity of the adjuvant itself affect the safety of the adjuvant. According to the level of IgG antibody in mice, the level of antibody in IgG positive group and CpG-sw group was the highest, and that in other groups was significantly higher than that in negative group. The concentration of cytokines in the CpG group was slightly higher than that in the negative group, but the increase was not obvious, and the concentration of cytokines in each oil adjuvant group was lower than that in the CpG group. However, the decrease was not obvious. Different adjuvants also showed different responses to T T B lymphocytes. The characteristics of these typical adjuvants are different in dosage form, water solubility and immune mechanism, showing different ways, trends and characteristics of immune enhancement.
【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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