本文選題：核酸疫苗　切入點(diǎn)：乙肝治療性DNA疫苗　出處：《第二軍醫(yī)大學(xué)》2006年博士論文　論文類型：學(xué)位論文

【摘要】：核酸疫苗(DNA疫苗)是指將含有編碼抗原基因的真核表達(dá)載體直接接種體內(nèi),在體內(nèi)表達(dá)相應(yīng)抗原刺激機(jī)體產(chǎn)生針對(duì)該抗原的免疫應(yīng)答,產(chǎn)生保護(hù)性免疫。核酸疫苗由于其獨(dú)特的雙免疫激活作用而受到越來(lái)越多的關(guān)注,有一些核酸疫苗已經(jīng)進(jìn)而Ⅱ期、Ⅲ期臨床研究,然而,核酸疫苗的免疫效果卻始終不甚理想,在動(dòng)物實(shí)驗(yàn)中,對(duì)于猴子、猩猩等大動(dòng)物,疫苗用量過(guò)大,為此,探索增強(qiáng)核酸疫苗免疫效果及降低疫苗用量的策略研究是十分必要的。 劑型改造是提高DNA疫苗免疫效果的方法之一。本課題中探討了一種新型的DNA疫苗給藥系統(tǒng),用聚乳酸/羥基乙酸共聚物(PLGA)包裹DNA疫苗形成微球疫苗后免疫小鼠,觀察免疫后小鼠體液免疫、細(xì)胞免疫及粘膜免疫的效果。實(shí)驗(yàn)中,以乙肝治療性DNA疫苗和多表位口蹄疫DNA疫苗為候選疫苗,應(yīng)用PLGA包裹形成微球疫苗,首先在體外實(shí)驗(yàn)中驗(yàn)證了疫苗微球?qū)乖f呈細(xì)胞的靶向作用。小鼠巨噬細(xì)胞系Raw264.7細(xì)胞被用于體外吞噬實(shí)驗(yàn),模擬抗原遞呈細(xì)胞對(duì)疫苗抗原的攝取過(guò)程。通過(guò)姬姆薩染色,證實(shí)了微球疫苗的可吞噬性;熒光標(biāo)記的疫苗微球的應(yīng)用,可以更準(zhǔn)確的動(dòng)態(tài)觀察微球的吞噬過(guò)程,在吞噬的2小時(shí)、4小時(shí)、8小時(shí)后分別收集細(xì)胞熒光顯微鏡觀察,可以看到隨著時(shí)間延長(zhǎng),細(xì)胞內(nèi)點(diǎn)狀熒光逐漸融合,熒光強(qiáng)度逐漸增加,進(jìn)一步證實(shí)微球的可吞噬性。吞噬后的微球能否成功釋放疫苗,疫苗抗原能否正確表達(dá)?后續(xù)實(shí)驗(yàn)中,在體內(nèi)、體外兩個(gè)層面上證實(shí)了抗原成份在抗原遞呈細(xì)胞中的正確表達(dá)。當(dāng)應(yīng)用PLGA包裹的乙肝DNA疫苗口服免疫小鼠后,可全面的激發(fā)機(jī)體的細(xì)胞免疫和體液免疫,還可在局部引流淋巴結(jié)中觀察到疫苗抗原的表達(dá),觀察到體現(xiàn)粘膜免疫效果的IgA的分泌,充分證實(shí)了口服乙肝DNA疫苗良好的免疫效果,獲得了有應(yīng)用前景的口服治療性乙肝DNA疫苗。在多表位口蹄疫DNA疫苗的研究中發(fā)現(xiàn),PLGA包裹可以降低疫苗的免疫劑量、減少疫苗的免疫次數(shù),使新型口蹄疫DNA疫苗更廉價(jià)、使用更方便。 膜聯(lián)蛋白B1基因是本課題組以免疫篩選法從豬囊尾蚴cDNA文庫(kù)中得到的一個(gè)新基因,其基因產(chǎn)物是豬帶絳蟲囊蟲病的診斷用抗原,也是很好的保護(hù)性抗原,以annexin B1為抗原基礎(chǔ)構(gòu)建的囊蟲DNA疫苗,在豬的保護(hù)性實(shí)驗(yàn)中可保護(hù)仔豬免于蟲卵沖擊后的感染或提高減蟲率。對(duì)于一個(gè)高免疫原性的疫苗抗原,其結(jié)構(gòu)的解析、生物學(xué)活性的認(rèn)識(shí)是安全、有效的利用疫苗抗原的基礎(chǔ),為了找到能
[Abstract]:Nucleic acid vaccine (DNA vaccine) means that the eukaryotic expression vector containing the encoded antigen gene is directly inoculated into the body and the corresponding antigen is expressed in the body to stimulate the body to produce an immune response to the antigen. The nucleic acid vaccine has attracted more and more attention because of its unique double immune activation. Some nucleic acid vaccines have gone on to phase II and III clinical studies. However, the immune effect of nucleic acid vaccine has not been satisfactory. In animal experiments, the dosage of vaccine is too large for big animals such as monkeys and orangutans. Therefore, it is necessary to explore the strategy of enhancing the immune effect of nucleic acid vaccine and reducing the dosage of vaccine. Dosage form modification is one of the methods to improve the immune effect of DNA vaccine. In this paper, a new DNA vaccine delivery system was studied, in which mice were immunized with DNA vaccine encapsulated with polylactic acid / glycolic acid copolymer. To observe the effect of humoral immunity, cellular immunity and mucosal immunity of mice after immunization, the therapeutic DNA vaccine of hepatitis B and the DNA vaccine of multiepitope foot-and-mouth disease were used as candidate vaccines, and the microsphere vaccine was formed by PLGA encapsulation. The mouse macrophage cell line Raw264.7 cells were used for phagocytosis in vitro, mimicking the process of antigen uptake by antigen presenting cells. The results showed that the phagocytosis of the microsphere vaccine was confirmed, and the application of fluorescent labeled vaccine microspheres could more accurately observe the phagocytosis process of the microspheres, and the fluorescence microscope was collected after 2 hours and 4 hours and 8 hours of phagocytosis, respectively. It can be seen that as the time goes on, the intracellular dot fluorescence gradually fuses and the fluorescence intensity increases, which further proves the phagocytosis of the microspheres, and whether the phagocytic microspheres can release the vaccine successfully, and whether the vaccine antigen can be correctly expressed? In subsequent experiments, in vivo and in vitro, the correct expression of antigen components in antigen-presenting cells was confirmed. When mice were immunized orally with PLGA encapsulated hepatitis B DNA vaccine, the cellular and humoral immunity of the body could be fully stimulated. The expression of vaccine antigen was also observed in the local drainage lymph nodes, and the secretion of IgA, which reflected the mucosal immune effect, was observed, which fully confirmed the good immune effect of the oral hepatitis B DNA vaccine. In the study of multiepitope foot-and-mouth disease (FMD) DNA vaccine, we found that PLGA-encapsulated vaccine can reduce the dose of vaccine, reduce the number of times of immunization, and make the new DNA vaccine cheaper. It is more convenient to use. Annexin B1 gene is a new gene obtained from cDNA library of cysticercus cellulosae by immune screening method. Its gene product is the diagnostic antigen of Taenia solium cysticercosis, and it is also a good protective antigen. The cysticercosis DNA vaccine constructed on the basis of annexin B1 can protect piglets from infection or increase the rate of worm reduction in the protective experiment. For a vaccine antigen with high immunogenicity, its structure is analyzed. The understanding of biological activity is the basis for the safe and effective use of vaccine antigens.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

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相關(guān)期刊論文 前1條

1 孫樹漢,王俊霞,陳蕊雯,陸惠萍,彭郁蔥,王朝霞;囊蟲病診斷用抗原編碼cDNA的分子克隆[J];中國(guó)寄生蟲學(xué)與寄生蟲病雜志;1997年01期

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本文編號(hào)：1586915