【摘要】：Quil A是從南美Quillaja皂莫利納的樹皮中提取的三萜皂苷，通過高效液相色譜分析包含至少5種不同的皂苷，是一種表面活性劑，易溶于水、穩(wěn)定、表面活性較好，是具有輔助屬性的皂苷。1936年，第一次作為佐劑由Thibaulu和Richou部署在獸用疫苗，，隨之被發(fā)展起來。PICKCa是一種改進的佐劑，是復(fù)雜的基于聚肌胞核苷酸制定的，聚肌胞本身對人體有毒性作用，但同時也具有強烈的佐劑作用，卡那霉素和氯化鈣能迅速水解于其中。作為一個帶正電的分子，卡那霉素能夠中和聚肌胞的負(fù)電荷，使聚肌胞更穩(wěn)定，抗水解。添加氯化鈣則有利于聚肌胞進入細(xì)胞�？傊�，PICKCa是一種穩(wěn)定安全的佐劑。 探討QuilA、PICKCa及二者聯(lián)合使用對實驗動物免疫應(yīng)答的調(diào)節(jié)作用，分別給小鼠、豬注射QuilA、PICKCa及二者混合物，于注射后不同時間采血，檢測細(xì)胞因子水平；注射后第7d檢測豬T淋巴細(xì)胞的體外增殖反應(yīng)。實驗結(jié)果表明小鼠和豬在注射藥物之后均未出現(xiàn)任何不良反應(yīng)；實驗組動物的IFN-γ、IL-2、IL-4、IL-10水平均顯著高于對照組；豬T淋巴細(xì)胞體外增殖能力也顯著高于對照組。說明QuilA、PICKCa對小鼠和豬具有良好的免疫調(diào)節(jié)作用，是具有良好應(yīng)用前景的免疫佐劑。 為研究Quil A對O型口蹄疫DNA疫苗的免疫增強作用，在FMD核酸疫苗pVIRIL18P1中加入Quil A，經(jīng)肌肉注射免疫豬，檢測豬VP1結(jié)構(gòu)蛋白抗體及細(xì)胞因子水平，觀察T淋巴細(xì)胞增殖情況，并與無佐劑組進行比較。結(jié)果表明，首免用豬口蹄疫核酸疫苗pVIRIL18P1及二免用重組雞痘疫苗vUTAL3CP1中加入Quil A，兩次免疫后抗體水平、細(xì)胞因子水平及T淋巴細(xì)胞增殖率都顯著升高，與空白對照組比較差異顯著（P0.05）甚至極顯著(P0.01)，與未加佐劑組及商品化疫苗比較有顯著差異。表明Quil A對O型口蹄疫DNA疫苗具有較好的免疫增強作用。 為研究PICKCa對豬繁殖與呼吸綜合征和豬2型圓環(huán)病毒病二聯(lián)核酸疫苗的免疫增強效果，在二聯(lián)核酸疫苗pIRES-ORF2-ORF5m-ORF6a中加入PICKCa，經(jīng)肌肉注射免疫豬，檢測豬繁殖與呼吸綜合征和豬2型圓環(huán)病毒病病毒抗體及細(xì)胞因子水平，觀察T淋巴細(xì)胞增殖情況，并與無佐劑組進行比較。豬體免疫試驗結(jié)果證明，構(gòu)建的重組核酸疫苗加以PICKCa均能刺激實驗豬產(chǎn)生PRRSV、PCV2抗體。細(xì)胞免疫檢測結(jié)果表明PRRSV、PCV2特異性T淋巴細(xì)胞增殖、IFN-γ和IL-2分泌水平顯著提高、CTL殺傷活性增強，表明其能夠介導(dǎo)較好的Th1類免疫反應(yīng)。重組核酸疫苗免疫豬的組織器官無病理損傷，可以防止豬體病毒血癥的出現(xiàn)。攻毒保護實驗證明，添加PICKCa佐劑的重組核酸疫苗對實驗豬起到明顯的免疫保護提升作用。
[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- 緯 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- 緯 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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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