本文選題：佐劑　切入點(diǎn)：免疫增強(qiáng)　出處：《中國(guó)海洋大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：疫苗免疫是現(xiàn)今預(yù)防動(dòng)物傳染病的最有效的手段之一。動(dòng)物疫苗的生產(chǎn)研究具有重大的社會(huì)經(jīng)濟(jì)效益。佐劑作為一種免疫增強(qiáng)劑，成為絕大部分疫苗不可或缺的一部分。新型佐劑也隨著疫苗市場(chǎng)的拓展，具有廣闊的前景。 國(guó)內(nèi)佐劑研究不斷深入，有研究驗(yàn)證了一些常規(guī)佐劑在亞單位疫苗中的應(yīng)用效果，但自主研發(fā)的商品化佐劑較少，很多動(dòng)物疫苗的生產(chǎn)依賴進(jìn)口佐劑。傳統(tǒng)的乳化方法，增加了企業(yè)能耗，使得成本過(guò)高。 本研究就是基于生產(chǎn)所需的高安全性、高免疫增強(qiáng)性、低能耗、低成本的要求，設(shè)計(jì)研發(fā)了一組油乳劑佐劑和一款高通量制備的富含豬特異性CpG的佐劑，以期開(kāi)發(fā)適合豬口蹄疫亞單位疫苗的商品化佐劑。 本研究主要采用了HLB法對(duì)復(fù)合乳劑形成的各組配方進(jìn)行篩選，確定最佳方案；在制備微乳劑佐劑時(shí)主要通過(guò)三元相圖法繪制微乳區(qū)域來(lái)尋求最優(yōu)配伍。 復(fù)合油佐劑COA，為一款由更安全的油相成分和毒性較小的乳化劑混合而成的油佐劑，其乳化工藝及乳劑性質(zhì)類似法國(guó)Seppic生產(chǎn)的ISA206佐劑，低速攪拌即可乳化；W/O/W劑型使乳劑即時(shí)、持續(xù)釋放抗原，用以迅速持久的免疫。 MOM佐劑，為一款礦物油微乳佐劑，為自乳化體系。油水相混合均勻，即可形成納米級(jí)微乳，穩(wěn)定性極高。W/O劑型和極小顆粒有利于抗原的緩慢釋放，提高免疫。 MFM佐劑，為一款用可代謝動(dòng)物油配制的微乳佐劑。作為MF59佐劑的改進(jìn)配方，它同樣是自乳化體系，油水相混勻即可乳化，乳劑顆粒達(dá)到納米級(jí)，穩(wěn)定性極高。 以上3款油佐劑根據(jù)商品化佐劑即開(kāi)即用的需求設(shè)計(jì)，乳化簡(jiǎn)單，穩(wěn)定性強(qiáng)。 CpG-sw佐劑，為一款通過(guò)基因工程手段，設(shè)計(jì)制備的富含豬特異性CpG基序的核酸佐劑，同時(shí)借助工程菌增殖提高CpG生產(chǎn)通量，提升了CpG的可利用度。 本研究將試制的幾款佐劑合并胸腺素，形成制劑類、核酸類、蛋白類三類典型的佐劑系統(tǒng)，以傳統(tǒng)白油佐劑作為陽(yáng)性對(duì)照，無(wú)佐劑抗原作為陰性對(duì)照分別與亞單位疫苗制成佐劑疫苗，免疫小鼠，經(jīng)體內(nèi)實(shí)驗(yàn)，以初步篩選出合適的亞單位疫苗佐劑。 經(jīng)過(guò)動(dòng)物實(shí)驗(yàn)驗(yàn)證，發(fā)現(xiàn)MOM組、白油陽(yáng)性組和CpG-sw組小鼠在免疫初期出現(xiàn)全身反應(yīng)，1~2日內(nèi)反應(yīng)消失。MOM組和白油陽(yáng)性組在注射部位產(chǎn)生硬結(jié)，難以消化，MOM硬結(jié)較小。其他組未見(jiàn)反應(yīng)。制劑外相的親水性和佐劑本身的毒性影響到佐劑的安全性。 從小鼠IgG抗體水平來(lái)看，MOM組、白油陽(yáng)性組和CpG-sw組抗體水平最高，其他各組較陰性組也有明顯提高，發(fā)揮了佐劑作用。從脾細(xì)胞細(xì)胞因子濃度來(lái)看，CpG組、Tα1組細(xì)胞因子濃度較陰性輕微提高，但提高不明顯，各油佐劑組均有下降，但下降不明顯。不同佐劑在刺激T、B淋巴細(xì)胞反應(yīng)方面也表現(xiàn)不一。 這幾款典型佐劑特點(diǎn)各異，在劑型、水溶性、免疫機(jī)制方面都有不同，體現(xiàn)出不同免疫增強(qiáng)的途徑、趨勢(shì)和特點(diǎn)。
[Abstract]:Vaccine immunization is one of the most effective methods to prevent animal infectious diseases. Research on animal vaccine production has great social and economic benefits. As an indispensable part of most vaccines, new adjuvants have broad prospects with the development of vaccine market. The domestic research on adjuvant has been deepening. Some studies have verified the application effect of some conventional adjuvants in subunit vaccines, but the commercial adjuvants developed by ourselves are relatively few. Many animal vaccines are produced by imported adjuvants. Traditional emulsification methods. Increases the enterprise energy consumption, causes the cost to be too high. Based on the requirements of high safety, high immunity enhancement, low energy consumption and low cost for production, a group of oil emulsion adjuvants and a high-throughput adjuvant rich in porcine specific CpG were designed and developed. To develop a commercial adjuvant for porcine foot-and-mouth disease subunit vaccine. In this study, the HLB method was used to screen the formulations of the composite emulsion and to determine the best formula, and the ternary phase diagram method was used to draw the region of the microemulsion to find the best compatibility in the preparation of the microemulsion adjuvant. The compound oil adjuvant, COA, is an oil adjuvant made from a safer oil phase and a less toxic emulsifier. Its emulsifying process and emulsion properties are similar to those of the ISA206 adjuvant produced by Seppic in France. The emulsion can be emulsified by low speed stirring in the form of W / O / W / W. The persistent release of antigens is used for rapid and persistent immunity. MOM adjuvant is a kind of mineral oil microemulsion adjuvant, which is self-emulsifying system. When the oil and water are mixed evenly, the nanometer microemulsion can be formed. MFM adjuvant is a microemulsion adjuvant prepared from metabolizable animal oil. As an improved formula of MF59 adjuvant, it is also a self-emulsifying system. The above three oil adjuvants are designed according to the requirements of commercial adjuvants, easy emulsification and strong stability. CpG-sw adjuvant is a kind of nucleic acid adjuvant which is rich in porcine specific CpG motifs designed and prepared by genetic engineering method. At the same time, the production flux of CpG is increased with the help of the multiplication of engineering bacteria, and the availability of CpG is improved. In this study, several adjuvants were combined with thymosin to form three typical adjuvant systems: preparation, nucleic acid and protein. The traditional white oil adjuvant was used as positive control. Adjuvant free antigen was used as negative control to make adjuvant vaccine with subunit vaccine respectively. The mice were immunized with adjuvant antigen. The suitable adjuvant of subunit vaccine was screened in vivo. After animal experiment, it was found that the systemic reaction disappeared within 1 ~ 2nd in MOM group, white oil positive group and CpG-sw group. The other groups have no reaction. The hydrophilicity of the preparation and the toxicity of the adjuvant itself affect the safety of the adjuvant. According to the level of IgG antibody in mice, the level of antibody in IgG positive group and CpG-sw group was the highest, and that in other groups was significantly higher than that in negative group. The concentration of cytokines in the CpG group was slightly higher than that in the negative group, but the increase was not obvious, and the concentration of cytokines in each oil adjuvant group was lower than that in the CpG group. However, the decrease was not obvious. Different adjuvants also showed different responses to T T B lymphocytes. The characteristics of these typical adjuvants are different in dosage form, water solubility and immune mechanism, showing different ways, trends and characteristics of immune enhancement.
【學(xué)位授予單位】：中國(guó)海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 胡金家,汪華僑,曲懷剛,徐杰,姚志彬;Aβ_(42)及亞單位疫苗誘導(dǎo)小鼠抗體產(chǎn)生及其中和Aβ_(42)的細(xì)胞毒性[J];細(xì)胞與分子免疫學(xué)雜志;2004年02期

2 蔣文俊,李華春,李樂(lè),信愛(ài)國(guó),廖德芳;口蹄疫疫苗研究進(jìn)展[J];畜牧與獸醫(yī);2004年05期

3 景志忠;蒙學(xué)蓮;王佩雅;竇永喜;李輝;駱學(xué)農(nóng);鄭亞?wèn)|;才學(xué)鵬;;CpG DNA重組質(zhì)粒對(duì)豬囊尾蚴抗原的免疫佐劑效應(yīng)研究[J];中國(guó)寄生蟲學(xué)與寄生蟲病雜志;2006年02期

4 徐云龍;徐盛;楊欣欣;;溶菌酶脂質(zhì)體的制備工藝[J];華東理工大學(xué)學(xué)報(bào);2006年08期

5 郭愛(ài)珍,唐泰山,陸承平;細(xì)菌CpG-DNA對(duì)犬免疫增強(qiáng)效果的研究[J];中國(guó)獸醫(yī)科技;2001年12期

6 張健;禽用油乳劑滅活苗乳化技術(shù)[J];天津農(nóng)業(yè)科學(xué);1998年S1期

7 周永東,張明程,蔣琳,沈心亮,畢勝利;粘膜免疫戊型肝炎試驗(yàn)性疫苗的研究[J];病毒學(xué)報(bào);2005年01期

8 馬文芝,劉湘,朱慶輝;雞油乳劑滅活苗佐劑乳化方法與乳化效果[J];動(dòng)物科學(xué)與動(dòng)物醫(yī)學(xué);1998年01期

9 周紅蕾;李春玲;王貴平;侯加法;;脂質(zhì)體作為疫苗免疫佐劑的應(yīng)用研究進(jìn)展[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2006年02期

10 戴愛(ài)玲;李曉華;;雞油乳劑苗乳化工藝的探討[J];龍巖學(xué)院學(xué)報(bào);2005年06期

，

本文編號(hào)：1590994