Quil A、PICKCa对FMDV、PCV2-PRRSV DNA疫苗免疫增强作用的效果观察

发布时间：2019-04-09 13:28

【摘要】：Quil A是从南美Quillaja皂莫利纳的树皮中提取的三萜皂苷，通过高效液相色谱分析包含至少5种不同的皂苷，是一种表面活性剂，易溶于水、稳定、表面活性较好，是具有辅助属性的皂苷。1936年，第一次作为佐剂由Thibaulu和Richou部署在兽用疫苗，，随之被发展起来。PICKCa是一种改进的佐剂，是复杂的基于聚肌胞核苷酸制定的，聚肌胞本身对人体有毒性作用，但同时也具有强烈的佐剂作用，卡那霉素和氯化钙能迅速水解于其中。作为一个带正电的分子，卡那霉素能够中和聚肌胞的负电荷，使聚肌胞更稳定，抗水解。添加氯化钙则有利于聚肌胞进入细胞。总之，PICKCa是一种稳定安全的佐剂。探讨QuilA、PICKCa及二者联合使用对实验动物免疫应答的调节作用，分别给小鼠、猪注射QuilA、PICKCa及二者混合物，于注射后不同时间采血，检测细胞因子水平；注射后第7d检测猪T淋巴细胞的体外增殖反应。实验结果表明小鼠和猪在注射药物之后均未出现任何不良反应；实验组动物的IFN-γ、IL-2、IL-4、IL-10水平均显著高于对照组；猪T淋巴细胞体外增殖能力也显著高于对照组。说明QuilA、PICKCa对小鼠和猪具有良好的免疫调节作用，是具有良好应用前景的免疫佐剂。为研究Quil A对O型口蹄疫DNA疫苗的免疫增强作用，在FMD核酸疫苗pVIRIL18P1中加入Quil A，经肌肉注射免疫猪，检测猪VP1结构蛋白抗体及细胞因子水平，观察T淋巴细胞增殖情况，并与无佐剂组进行比较。结果表明，首免用猪口蹄疫核酸疫苗pVIRIL18P1及二免用重组鸡痘疫苗vUTAL3CP1中加入Quil A，两次免疫后抗体水平、细胞因子水平及T淋巴细胞增殖率都显著升高，与空白对照组比较差异显著（P0.05）甚至极显著(P0.01)，与未加佐剂组及商品化疫苗比较有显著差异。表明Quil A对O型口蹄疫DNA疫苗具有较好的免疫增强作用。为研究PICKCa对猪繁殖与呼吸综合征和猪2型圆环病毒病二联核酸疫苗的免疫增强效果，在二联核酸疫苗pIRES-ORF2-ORF5m-ORF6a中加入PICKCa，经肌肉注射免疫猪，检测猪繁殖与呼吸综合征和猪2型圆环病毒病病毒抗体及细胞因子水平，观察T淋巴细胞增殖情况，并与无佐剂组进行比较。猪体免疫试验结果证明，构建的重组核酸疫苗加以PICKCa均能刺激实验猪产生PRRSV、PCV2抗体。细胞免疫检测结果表明PRRSV、PCV2特异性T淋巴细胞增殖、IFN-γ和IL-2分泌水平显著提高、CTL杀伤活性增强，表明其能够介导较好的Th1类免疫反应。重组核酸疫苗免疫猪的组织器官无病理损伤，可以防止猪体病毒血症的出现。攻毒保护实验证明，添加PICKCa佐剂的重组核酸疫苗对实验猪起到明显的免疫保护提升作用。
[Abstract]:Quil A is a triterpenoid saponins extracted from the bark of Quillaja soap Molina, South America. It contains at least five different saponins by high performance liquid chromatography (HPLC). It is a surfactant, soluble in water, stable and has good surface activity. Saponins with auxiliary properties. In 1936, they were first deployed as adjuvants in veterinary vaccines by Thibaulu and Richou. PICKCA is an improved adjuvant that is complex based on polynucleotides. The polymyocytes themselves have toxic effects on human body, but also have a strong adjuvant effect. Kanamycin and calcium chloride can be hydrolyzed rapidly. As a positively charged molecule, kanamycin can neutralize the negative charge of the polymyocytes, making the polymyocytes more stable and anti-hydrolysis. The addition of calcium chloride was beneficial to polymyocytes entering the cells. In short, PICKCa is a stable and safe adjuvant. To explore the regulatory effect of QuilA,PICKCa and its combination on immune response of experimental animals, QuilA,PICKCa and its mixture were injected into mice and pigs respectively. Blood samples were collected at different time after injection to detect cytokine level. The proliferation of porcine T lymphocytes in vitro was detected on the 7th day after injection. The results showed that there were no adverse reactions in mice and pigs after injection of drugs, and the levels of IFN- 纬 and IL-2,IL-4,IL-10 in the experimental group were significantly higher than those in the control group. The proliferation ability of porcine T lymphocytes in vitro was also significantly higher than that of the control group. It is concluded that QuilA,PICKCa has a good immunomodulatory effect on mice and pigs, and it is a promising immunoadjuvant. In order to study the immunoenhancement effect of Quil A on DNA vaccine of type O foot-and-mouth disease (FMD), Quil A was added to FMD nucleic acid vaccine pVIRIL18P1 to immunize pigs by intramuscular injection to detect the level of VP1 structural protein antibody and cytokines, and to observe the proliferation of T lymphocytes. And compared with the non-adjuvant group. The results showed that the level of antibody, the level of cytokines and the proliferation rate of T lymphocytes were significantly increased after two times of immunization with the addition of Quil A into the nucleic acid vaccine pVIRIL18P1 and the recombinant fowlpox vaccine vUTAL3CP1 for the first and second immunization of swine foot-and-mouth disease (FMD), respectively. Compared with the blank control group, the difference was significant (P0.05) or extremely significant (P0.01), and significantly different from the non-adjuvant group and commercial vaccine. The results showed that Quil A had a better immune enhancement effect on DNA vaccine of type O foot-and-mouth disease (FMD). In order to study the immunoenhancement effect of PICKCa on porcine reproductive and respiratory syndrome (PRRS) and porcine circovirus type 2 (PCV) vaccine, PICKCa, was added to the pIRES-ORF2-ORF5m-ORF6a vaccine to immunize pigs by intramuscular injection. The antibody and cytokine levels of porcine reproductive and respiratory syndrome (PRRS) and porcine circovirus type 2 virus (PRV-2) were detected. The proliferation of T lymphocytes was observed and compared with that of non-adjuvant group. The results of porcine immunity test showed that the recombinant nucleic acid vaccine combined with PICKCa could stimulate the production of PRRSV,PCV2 antibody in experimental pigs. The results of cellular immunoassays showed that PRRSV,PCV2-specific T lymphocytes proliferated, the secretion of IFN- 纬 and IL-2 increased significantly, and the killing activity of CTL increased, which indicated that it could mediate better Th1-like immune response. Recombinant nucleic acid vaccine vaccinated swine tissues and organs without pathological damage, can prevent the occurrence of swine viremia. The experimental results showed that the recombinant nucleic acid vaccine supplemented with PICKCa adjuvant could significantly enhance the immune protection of experimental pigs.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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