中药疫苗稀释剂提高猪伪狂犬和猪瘟弱毒疫苗的免疫效果研究

发布时间：2018-08-13 09:27

【摘要】：传染病是威胁养猪业健康发展的危害因素之一。伪狂犬病(Pseudorabies,Pr)是由伪狂犬病毒(Pseudorabies virus,PrV))引起的一种高度接触性动物传染病。Pr对养猪业的危害很大,15日龄以内仔猪感染后死亡率高达100%,断奶仔猪发病后死亡率约10～20%。猪瘟(Classical swine fever,CSF)是由猪瘟病毒(Classical swine fever virus,CSFV)引起的另一种烈性传染病,对我国养猪业造成重大经济损失。目前我国广泛采用弱毒疫苗接种的方法来控制Pr和CSF,疫苗使用前一般须要经过生理盐水稀释才能注射动物。但在很多情况下疫苗不能有效地激发机体产生理想的免疫应答而导致免疫失败。因此,本论文拟研究具有免疫增强作用的疫苗稀释剂以提高伪狂犬弱毒(attenuated pseudorabies virus,aPrV)疫苗和猪瘟弱毒疫苗的免疫效果,为临床应用提供参考。1.疫苗稀释剂配方的研究目的:筛选合适的aPrV疫苗免疫增强剂配方。方法:以ICR小鼠为模型,免疫aPrV疫苗,以血清特异性PrV gB抗体为指标,评价白术多糖(RAMPS)和人参茎叶皂苷(GSLS)的免疫增强作用;以血清PrVgB、PrVgBIgGl、PrVgBIgG2a和攻毒保护为指标,评价候选稀释剂配方的免疫增强效果。结果:GSLS、RAMPS和硫柳汞(TS)均能增强小鼠血清PrVgB抗体水平,以GSLS的免疫增强作用最好。GSLS联合RAMPS没有协同作用,GSLS联合TS表现出协同作用,以GSLS-TS(6μg-1μg)的免疫增强效果最为显著。该配方能提前促进血清PrV gB、PrV gB IgG1和PrV gB IgG2a抗体的产生,并能在较长时间维持抗体在较高水平。伪狂犬野毒攻毒后,含GSLS-TS的稀释剂疫苗组小鼠的存活率明显高于生理盐水稀释疫苗组(70%vs40%)。2.灭菌方法和贮存条件对GSLS-TS免疫活性的影响目的:观察灭菌方法和贮存条件对GSLS-TS免疫增强作用的影响。方法:以ICR小鼠为模型,分别用不同灭菌方法处理的GSLS溶液和不同贮存条件下保存1年的GSLS-TS溶液稀释aPrV疫苗后免疫小鼠,以血清特异性抗体水平为指标,评价灭菌方法和贮存条件对GSLS-TS免疫增强作用的影响。结果:高温(100～121℃)处理和光照均会显著降低GSLS-TS的免疫增强效果。因此,GSLS-TS溶液不宜高温灭菌,并适合在低温避光条件下保存。3.GSLS-TS和其它6种商品稀释剂佐剂作用的比较目的:将GSLS-TS溶液与6个厂家的aPrV疫苗专用稀释剂进行效果对比。方法:以ICR小鼠为模型,用GSLS-TS溶液稀释6个不同厂家的aPrV疫苗,并与它们各自的专用疫苗稀释剂和生理盐水进行对比,以血清特异性抗体水平为指标。结果:除“晶制”厂家的稀释剂外,其它厂家的疫苗稀释剂免疫增强作用不明显,与生理盐水相比没有显著的差异。GSLS-TS溶液的免疫增强作用显著优于生理盐水和6种专用疫苗稀释剂。4.GSLS-TS提高猪对Pr和CSF弱毒疫苗的抗体反应研究目的:通过本动物试验观察GSLS-TS对猪弱毒疫苗的免疫增强作用。方法:用GSLS-TS溶液分别稀释Pr和CSF弱毒疫苗后免疫猪,以血清特异性抗体水平为指标,评价GSLS-TS对疫苗诱导体液免疫的影响。结果:GSLS-TS能同时促进猪对Pr和CSF两种弱毒疫苗的体液免疫反应,显著促进血清特异性抗体、抗体亚类和中和抗体滴度。5.GSLS-TS对细胞免疫的影响目的:评价GSLS-TS对疫苗诱导的细胞免疫的影响。方法:用GSLS-TS溶液稀释aPrV疫苗后免疫小鼠,24h后检测脾脏中NK细胞的杀伤活性、血清中IFN-γ水平;二免二周时,检测脾淋巴细胞增殖活性和淋巴细胞中Thl和Th2类细胞因子分泌。结果:GSLS-TS能快速促进疫苗诱导的机体早期免疫反应(NK细胞杀伤活性、血清IFN-γ产生),显著增强脾脏淋巴细胞增殖活性,提高淋巴细胞分泌Thl(IFN-γ、IL-12)和Th2(IL-5、IL-10)类细胞因子水平。6.GSLS-TS免疫增强作用机制研究目的:初步探讨GSLS-TS免疫增强作用的机制。方法:将GSLS和(或)TS与小鼠巨噬细胞系 RAW264.7 共孵育,以miR-132、miR-146a、miR-147 和 miR-155 的mRNA表达为指标,分析GSLS或(和)TS对RAW264.7细胞中这4种miRs水平的影响;给ICR和C3H/HeJ小鼠免疫aPrV疫苗,以血清特异性抗体、淋巴细胞增殖反应以及淋巴细胞表达Thl和Th2类细胞因子mRNA的水平为指标,初步分析GSLS-TS免疫增强作用与TLR4的关系。结果:用GSLS或(和)TS与RAW264.7细胞共同培养时能显著提高细胞中 miR-132、miR-146a、miR-147 和 miR-155 的 mRNA 表达,而且 GSLS和TS表现出协同作用;GSLS-TS能同时提高ICR小鼠和C3H/HeJ小鼠免疫aPrV疫苗后血清特异性抗体反应,同时增强Con A和PrV抗原诱导的淋巴细胞增殖活性以及淋巴细胞表达Thl(IFN-γ、IL-12)和Th2(IL-5、IL-10)类细胞因子的mRNA水平,说明GSLS-TS的免疫增强作用不主要与TLR4相关。综上所述,GSLS和TS对aPrV疫苗诱导的抗体反应具有协同增强作用,GSLS-TS可显著增强aPrV免疫小鼠早期免疫反应、体液免疫反应、细胞免疫反应以及对野毒攻毒的抵抗力。本动物试验表明,稀释剂能显著增强猪对Pr和CSF弱毒疫苗诱导的抗体反应,显示出较好的临床应用潜力。GSLS-TS的免疫增强作用并不直接通过TLR4所介导,可能与免疫细胞中miR-132、miR-146a、miR-147和miR-155的表达上调相关,关于稀释剂的具体作用机制有待深入研究。
[Abstract]:Pseudorabies (Pr) is a highly contagious animal disease caused by Pseudorabies virus (PrV). Pr is very harmful to the pig industry. The mortality rate of piglets within 15 days of age after infection is as high as 100%, and that of weaned piglets is about 10-10%. 20%. Classical swine fever (CSF) is another severe infectious disease caused by Classical swine fever virus (CSFV), which has caused great economic losses to the pig industry in China. Animals, however, in many cases vaccines can not effectively stimulate the body to produce an ideal immune response and lead to immune failure. Therefore, this paper intends to study vaccine diluents with immune enhancement effect to improve the immune effect of attenuated pseudorabies virus (aPrV) vaccine and attenuated swine fever vaccine for clinical application. Objective: To select the appropriate immune enhancer formula of aPrV vaccine. Methods: ICR mice were immunized with aPrV vaccine, and serum specific PrV gB antibody was used as the index to evaluate the immune enhancement effect of Atractylodes macrocephala polysaccharide (RAMPS) and ginseng stem and leaf saponin (GSLS), serum PrV gB, PrVgBIgGl, PrVgBIgG2a and attack. Results: GSLS, RAMPS and thiomersal (TS) could enhance the level of serum PrVgB antibody in mice. GSLS combined with RAMPS had no synergistic effect, GSLS combined with TS showed synergistic effect, and GSLS-TS (6ug-1ug) had the most obvious effect. This formula can promote the production of serum PrV gB, PrV gB IgG1 and PrV gB IgG2a antibodies in advance, and can maintain a higher level of antibodies for a longer time. AIM: To observe the effect of sterilization and storage conditions on immune enhancement of GSLS-TS. Methods: ICR mice were immunized by diluting aPrV vaccine with GSLS-TS solution treated by different sterilization methods and GSLS-TS solution preserved under different storage conditions for one year. Serum specific antibody level was used as an index to evaluate the immune enhancement. Results: High temperature (100-121 C) treatment and illumination significantly reduced the immune enhancement effect of GSLS-TS. Therefore, GSLS-TS solution is not suitable for high temperature sterilization, and is suitable for storage under low temperature and light-proof conditions. 3. GSLS-TS and other six commercial diluents adjuvant effect comparison purposes: Methods: ICR mice were used to dilute 6 different manufacturers'aPrV vaccines with GSLS-TS solution, and compared with their own special vaccine diluents and saline. Serum specific antibody levels were taken as indicators. Diluents, other manufacturers of vaccine diluent immune enhancement effect is not obvious, compared with normal saline has no significant difference. The immune enhancement effect of.GSLS-TS solution is significantly better than normal saline and 6 special vaccine thinner.4.GSLS-TS to enhance the antibody response of pigs to Pr and CSF attenuated vaccines. Objective: To observe GS through this animal experiment. The immune enhancement effect of LS-TS on swine attenuated vaccine. Methods: immunized pigs were diluted with GSLS-TS solution diluted with Pr and CSF attenuated vaccine respectively. The serum specific antibody level was used as an indicator to evaluate the effect of GSLS-TS on the humoral immunity induced by vaccine. Results: GSLS-TS can also promote the humoral immune response of pigs to two attenuated vaccines of Pr and CSF, and significantly promote the immune response. Serum specific antibodies, antibody subclasses and neutralizing antibody titers. Results: GSLS-TS could rapidly promote the early immune response induced by vaccine (NK cell killing activity, serum IFN-gamma production), significantly enhance the proliferative activity of splenic lymphocytes, and enhance the secretion of Thl (IFN-gamma, IL-12) and Th2 (IL-5, IL-10) cytokines by lymphocytes. Level. 6. GSLS-TS immunopotentiation mechanism: To explore the mechanism of GSLS-TS immunopotentiation. Methods: GSLS and/or TS were co-incubated with mouse macrophage RAW264.7, and the mRNA expression of microRNAs-132, microRNAs-146 a, microRNAs-147 and microRNAs-155 were used as indicators to analyze the effects of GSLS or (and) TS on the levels of these four microRNAs in RAW264.7 cells. Immune aPrV vaccine was given to ICR and C3H/HeJ mice. Serum specific antibodies, lymphocyte proliferation reaction and the expression of Thl and Th2 cytokine mRNA in lymphocytes were used as indicators to preliminarily analyze the relationship between GSLS-TS immunopotentiation and TLR4. Results: When cultured with GSLS or (and) TS and RAW264.7 cells, the expression of Mi-1 was significantly increased. GSLS-TS can simultaneously enhance the serum specific antibody response in ICR mice and C3H/HeJ mice immunized with aPrV vaccine, and enhance the lymphocyte proliferation activity induced by Con A and PrV antigens and the expression of Thl (IFN-gamma, IL-12) and Th2 (IL-5, IL-1) in lymphocytes. To sum up, GSLS and TS have synergistic effects on antibody response induced by aPrV vaccine. GSLS-TS can significantly enhance the early immune response, humoral immune response, cellular immune response and resistance to field virus attack in aPrV immunized mice. This animal experiment shows that thinner can significantly enhance pig's antibody response induced by Pr and CSF attenuated vaccine, showing a good clinical potential..GSLS-TS's immune enhancement is not directly mediated by TLR4, which may be related to the up regulation of miR-132, miR-146a, miR-147 and miR-155 in immune cells. The mechanism needs to be further studied.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.28

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