猪瘟口服疫苗保护剂筛选及免疫效果评价

发布时间：2018-06-10 21:17

本文选题：猪瘟疫苗 + 口服免疫　；参考：《上海海洋大学》2015年硕士论文

【摘要】：猪瘟(classical swine fever,CSF)是由猪瘟病毒(Classical swine fever virus,CSFV)引起的一种猪的高度接触性传染病。该病呈急性或慢性感染,在许多国家和地区均有发生,给养猪业造成极大的危害。目前,疫苗接种仍是控制猪瘟疫情的主要手段。现阶段我国使用的猪瘟疫苗主要有猪瘟兔化弱毒株、家兔脾淋组织苗、乳猪肾细胞苗等注射型疫苗。国外报道猪瘟疫苗口服免疫获得了很好的效果,经口服免疫,可减少对猪只的应激且省时省力。因此研究拟通过筛选理想的保护剂,制备可经口服途径免疫的猪瘟疫苗。本研究采用两种方法制备口服抗原:以猪瘟病毒疫苗株与重组LTRG蛋白及冻干保护剂配伍,制备冻干抗原;另外利用壳聚糖-海藻酸钠包埋重组CSFV E2蛋白,制备微囊抗原;两种抗原分别口服饲喂小鼠,以细胞免疫指标和体液免疫指标评价抗原的免疫效果,筛选理想的抗原保护剂。1蛋白抗原包埋及免疫活性检测构建CSFV E2基因的原核表达质粒并表达蛋白,经纯化后将其作为抗原,与黏膜佐剂蛋白LTRG共同包埋制备壳聚糖-海藻酸钠微球,然后口服免疫小鼠,检测其免疫效果。结果,包埋E2和LTRG蛋白组小鼠的血清Ig G和黏膜Ig A水平均显著高于对照组,高于E2蛋白包埋组,表明LTRG蛋白作为一种黏膜佐剂,增强了小鼠的免疫反应。此外,包埋E2和LTRG蛋白组小鼠的抗体水平显著高于E2+LTRG蛋白组,说明壳聚糖-海藻酸钠微球作为包埋载体,将抗原进行了有效的呈递,从而诱导小鼠产生免疫反应。2冻干保护剂筛选及免疫效果评价研究配比了不同组分的冻干保护剂,包括明胶、乳糖和甘氨酸,与黏膜佐剂LTRG蛋白、CSFV按比例混合,经优化冻干工艺后,制备了4种CSFV的冻干苗。口服免疫小鼠,测定血清Ig G和黏膜Ig A抗体效价。结果,含明胶和甘氨酸的试验2组和含明胶的试验4组经冻干后外观形态较好,水溶后呈现澄清状态;试验2组和试验4组的冻干苗经口服免疫,诱导小鼠产生的Ig G抗体水平极显著高于CSFV组、CSFV+LT组及其他试验组;二免7天后试验2组小鼠产生的Ig A抗体极显著高于其他组。而且,试验2组小鼠脾脏的IFN-γ和IL-4细胞因子的表达量也极显著高于其他组。表明,在冻干过程中,明胶和甘氨酸对猪瘟病毒起到了良好的保护作用,提高了猪瘟病毒疫苗经口服途径的免疫效果。利用壳聚糖-海藻酸钠包埋制备重组蛋白微球抗原,能诱导机体产生良好的体液免疫反应和细胞免疫应答,证实包埋蛋白抗原具有良好的免疫效果。配伍不同组分的猪瘟病毒疫苗保护剂,制备的冻干疫苗,能有效提高机体免疫抗体水平,证实制备的冻干保护剂配方对猪瘟病毒有较好保护作用。本研究为猪瘟疫苗冻干保护剂筛选和不同剂型疫苗的开发奠定了基础。
[Abstract]:Classical swine virus (CSFV) is a highly contagious disease of swine caused by classical swine fever virus (CSFV). The disease is acute or chronic infection, which occurs in many countries and regions, causing great harm to pig industry. At present, vaccination is still the main means to control the outbreak of swine fever. At present, the main vaccines used in China include attenuated strain of hog fever rabbit, splenic tissue vaccine of rabbit, kidney cell vaccine of suckling pig and so on. Oral immunization against swine fever vaccine has been reported in foreign countries. Oral immunization can reduce stress and save time and effort in pigs. Therefore, the aim of this study is to prepare an oral vaccine against classical swine fever (CSFV) by screening ideal protective agents. In this study, oral antigens were prepared by two methods: the recombinant LTRG protein and freeze-dried protective agent were mixed with CSFV vaccine strain to prepare freeze-dried antigen, and the recombinant CSFV E2 protein was encapsulated with chitosan sodium alginate to prepare microencapsulated antigen. Two kinds of antigens were fed orally to mice respectively. The immune effects of the two antigens were evaluated by cellular and humoral immune indexes. The prokaryotic expression plasmid of CSFV E2 gene was constructed by screening the ideal antigen protectant, 1 protein encapsulation and immunoassay, and the protein was purified and used as antigen. Chitosan-sodium alginate microspheres were prepared by embedding with mucosal adjuvant protein LTRG. The results showed that the levels of serum IgG and mucosal IgA in E2 and LTRG protein group were significantly higher than those in control group and E2 protein group, indicating that LTRG protein, as a mucosal adjuvant, enhanced the immune response of mice. In addition, the antibody levels of mice in E2 and LTRG protein groups were significantly higher than those in E2 LTRG protein group, indicating that chitosan and sodium alginate microspheres were used as entrapment carriers to present antigens effectively. In order to induce the immune response of mice, the screening of lyophilized protectants and the evaluation of immune effect were carried out. The lyophilized protectants of different components, including gelatin, lactose and glycine, were mixed with mucosal adjuvant LTRG protein in proportion to CSFV. Four kinds of CSFV freeze-dried seedlings were prepared after optimized freeze-drying process. Mice were immunized orally and the titers of serum IgG and mucosal IgA antibody were determined. The results showed that group 2 with gelatin and glycine and group 4 with gelatin had better appearance after freeze-drying and showed clear state after water solubilization, and the freeze-dried seedlings of test group 2 and test 4 were immunized orally. The level of IgG antibody induced in mice was significantly higher than that in CSFV LT group and other test groups, and the level of IgA antibody in group 2 was significantly higher than that in other groups after 7 days. Moreover, the expression of IFN- 纬 and IL-4 cytokines in spleen of experimental group 2 was significantly higher than that of other groups. The results showed that gelatin and glycine had a good protective effect on CSFV during freeze-drying, and the immune effect of CSFV vaccine was improved by oral route. The recombinant protein microsphere antigen was prepared by encapsulation of chitosan and sodium alginate, which could induce good humoral immune response and cellular immune response. It was proved that the encapsulated protein antigen had a good immune effect. The freeze-dried vaccine prepared with different components of CSFV vaccine could effectively improve the immune antibody level of the body. It was proved that the lyophilized protective agent formula had a better protective effect on CSFV. This study laid a foundation for the screening of freeze-dried protective agents for swine fever vaccine and the development of different dosage forms of vaccine.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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