猪瘟活疫苗佐剂的制备及其增强疫苗免疫效果评价

发布时间：2018-11-05 14:46

【摘要】：猪瘟是严重危害养猪业的一种病毒性传染病,注射猪瘟疫苗是预防猪瘟的重要方法。目前常用的猪瘟疫苗佐剂副作用较强,安全性和增强免疫效果有待提高,同时,还存在黏度高,注射使用不方便等问题。本文针对新发展的猪瘟活疫苗,研制安全性好、能够有效增强免疫效果、使用方便的猪瘟活疫苗新型佐剂,并对O/W乳剂型猪瘟活疫苗佐剂的安全性和增强免疫效果进行评价。对W/O/W乳剂型疫苗佐剂和聚合物凝胶佐剂的处方和制备工艺进行了初步研究。开展的主要研究工作如下:(1)采用高压均质技术制备了O/W乳剂型猪瘟活疫苗佐剂,采用响应面实验设计优化了猪瘟活疫苗佐剂的处方和制备工艺,并对其稳定性、粒径、多分散指数(PDI)及Zeta电位进行表征。优化处方和工艺制备的O/W型疫苗佐剂平均粒径为100.4 nm,PDI为0.147,Zeta电位为-28.7 mV,离心稳定性实验表明其稳定性良好。(2)进行了O/W乳剂型猪瘟活疫苗佐剂小鼠安全性实验。结果表明,注射佐剂组小鼠体重和生存状态与空白对照组相比较没有差异,没有出现由佐剂引起的死亡或者明显的局部和全身不良反应。两周后解剖小鼠,检查发现其注射部位及心、肝、脾、肺、肾均正常,没有中毒症状。表明研制的O/W型猪瘟活疫苗佐剂安全性良好。(3)将O/W乳剂型疫苗佐剂与猪瘟疫苗混合后,肌肉注射免疫小鼠,进行佐剂增强免疫效果评价。实验结果表明,佐剂组小鼠血清中抗猪瘟(CSFV)抗体水平与对照组有显著差异,抗体水平能够持续较长的时间,表明O/W乳剂型猪瘟活疫苗佐剂能够有效诱导机体产生体液免疫应答;佐剂组小鼠血清中IL-4、IL-6和IFN-γ的水平均有明显提高,表明该疫苗佐剂能够诱导机体产生细胞免疫应答。(4)采用高速分散结合高压匀质技术制备W/O/W乳剂型疫苗佐剂,对其处方和制备工艺进行了研究,通过筛选确定了乳化性能好的复合乳化剂和高压匀质制备工艺条件,优化制备的W/O/W乳剂型疫苗佐剂稳定性好,黏度适中。(5)开展了聚合物凝胶佐剂的处方和制备工艺研究,对聚合物凝胶基质、乳化剂、油相以及高压匀质工艺进行了筛选和优化,优化处方和工艺制备的聚合物凝胶佐剂平均粒径为221.6 nm,PDI为0.114,Zeta电位为-29.7 mV,稳定性良好。
[Abstract]:Swine fever (CSF) is a viral infectious disease which seriously endangers the swine industry. Injection of CSFV vaccine is an important method to prevent swine fever. At present, the adjuvant of classical swine fever vaccine has strong side effects, safety and immune enhancement effect need to be improved, at the same time, there are some problems such as high viscosity, inconvenient injection and so on. In this paper, a new adjuvant for swine fever vaccine is developed, which is safe, effective and easy to use. The safety and immune enhancement of O / W emulsion adjuvant of swine fever vaccine were evaluated. The formulation and preparation of W/O/W emulsion vaccine adjuvant and polymer gel adjuvant were studied. The main research works are as follows: (1) the O / W emulsion adjuvant of hog cholera vaccine was prepared by high pressure homogenization technique. The formulation and preparation process of live vaccine adjuvant of hog fever were optimized by response surface experiment, and the stability of the adjuvant was analyzed. Particle size, polydispersity index (PDI) and Zeta potential were characterized. The average particle size of the adjuvant prepared by optimized formulation and process is 100.4 nm,PDI, and the Zeta potential of the adjuvant is -28.7 mV,. Centrifugation stability test showed that the stability was good. (2) the safety test of adjuvant of O / W emulsion CSFV vaccine was carried out in mice. The results showed that there was no difference in body weight and survival status between the adjuvant group and the blank control group. There was no death caused by adjuvant or obvious local and systemic adverse reactions. Two weeks later, the mice were dissected and the injection site, heart, liver, spleen, lung and kidney were normal. The results showed that the adjuvant of O / W type swine fever vaccine was safe. (3) the adjuvant was injected intramuscularly to immunize mice with the adjuvant of O / W emulsion vaccine and swine fever vaccine, and the adjuvant was used to evaluate the effect of adjuvant. The results showed that the level of anti-CSFV (CSFV) antibody in the adjuvant group was significantly different from that in the control group, and the antibody level could last for a long time. The results showed that the adjuvant of O / W emulsion CSFV vaccine could induce humoral immune response effectively. The serum levels of IL-4,IL-6 and IFN- 纬 in the adjuvant group were significantly increased. The results showed that the adjuvant could induce cellular immune response. (4) W/O/W emulsion vaccine adjuvant was prepared by high speed dispersion and high pressure homogenization. The formulation and preparation process of the adjuvant were studied. The preparation conditions of composite emulsifier with good emulsifying performance and high pressure homogenization were determined by screening, and the adjuvant of W/O/W emulsion vaccine prepared by optimization was stable. (5) the formulation and preparation of polymer gel adjuvant were studied. The polymer gel matrix, emulsifier, oil phase and high pressure homogenization process were screened and optimized. The average particle size of the polymer gel adjuvant prepared by the optimized formulation and process was 221.6 nm,PDI and the Zeta potential was -29.7 mV,. The average particle size of the polymer gel adjuvant was -29.7 mV,.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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