結(jié)核桿菌和布魯氏桿菌多價(jià)DNA疫苗的研究
發(fā)布時(shí)間：2018-09-14 07:33
【摘要】： 結(jié)核病和布魯氏病是嚴(yán)重危害人類健康和畜牧業(yè)發(fā)展的人畜共患傳染病。疫苗是控制結(jié)核病和布魯氏病的有效方法。目前,BCG是預(yù)防結(jié)核病的唯一疫苗,但其免疫效力不穩(wěn)定,且對成年人無保護(hù);S19疫苗是在牛布魯氏病中使用的疫苗,但血清學(xué)反應(yīng)強(qiáng),且安全性受到質(zhì)疑,有感染人的可能。因此,高效的新型疫苗對于控制結(jié)核病和布魯氏病蔓延至關(guān)重要。本論文在闡述結(jié)核病和布魯氏病疫苗研究的基礎(chǔ)上,著重分析了DNA疫苗的作用機(jī)制、發(fā)展現(xiàn)狀及其改進(jìn)策略。 用分子生物學(xué)的方法分別構(gòu)建了編碼結(jié)核桿菌三種抗原(Ag85B,MPT64和MPT83)和布魯氏桿菌三種抗原(BCSP31,SOD,和L7/L12)的原核表達(dá)載體,誘導(dǎo)表達(dá)并純化后得到抗原蛋白。構(gòu)建此六種抗原的真核表達(dá)載體,進(jìn)行體外瞬時(shí)表達(dá)實(shí)驗(yàn),用GFP偶聯(lián)和RT-PCR等方法確定蛋白表達(dá)的部位和表達(dá)量。大量提取制備真核質(zhì)粒作為DNA疫苗,免疫小鼠三次后8周,用高劑量的結(jié)核桿菌或布魯氏桿菌感染小鼠或大動物牛。本文從體液免疫水平、細(xì)胞因子水平、分泌細(xì)胞因子細(xì)胞的數(shù)量、T細(xì)胞激活比例、T細(xì)胞亞型的變化、殺傷性T細(xì)胞的定量、結(jié)合動物感染后臟器載菌量等多個(gè)指標(biāo),對各個(gè)組合的疫苗的優(yōu)勢及所誘導(dǎo)免疫反應(yīng)的進(jìn)行綜合評價(jià),以期對DNA疫苗效力及其佐劑觸發(fā)免疫反應(yīng)的機(jī)制進(jìn)行詮釋。實(shí)驗(yàn)結(jié)果表明,多價(jià)DNA疫苗能夠誘導(dǎo)更強(qiáng)的TH1型免疫反應(yīng),擴(kuò)大保護(hù)范圍,保護(hù)效力顯著高于單價(jià)DNA疫苗。 通過對比聯(lián)合疫苗與不同礦物質(zhì)佐劑、基因佐劑共同免疫后所產(chǎn)生的保護(hù)效果,證實(shí)佐劑基因佐劑IL-2、IL-12、IL-15、和無機(jī)佐劑DDA在誘導(dǎo)TH1型細(xì)胞反應(yīng),增強(qiáng)殺傷性T細(xì)胞的功能方面發(fā)揮重要作用。尤其是用六種抗原聯(lián)合DNA疫苗與IL-12共同免疫,產(chǎn)生了“一針治兩病”的效果,保護(hù)效率顯著高于目前使用的BCG和S19傳統(tǒng)疫苗。IL-15佐劑顯著提高了分泌IFN-γ的CD8+T細(xì)胞的數(shù)量,從體內(nèi)、體外實(shí)驗(yàn)證實(shí)了CD8+T細(xì)胞在對抗布魯氏桿菌過程中的重要作用。多肽佐劑KLKL5KLK提高了APC細(xì)胞對抗原的攝取、加工和遞呈能力,延長了免疫保護(hù)期、增強(qiáng)CD4+T細(xì)胞作用和細(xì)胞殺傷水平,為臨床應(yīng)用提供了重要證據(jù)。 用包裹劑PLGA達(dá)到DNA緩釋和避免核酸酶對DNA降解的效果以減少免疫劑量,結(jié)果表明PLGA在提高抗原表達(dá)量,延長表達(dá)時(shí)間上有顯著作用,在免疫攻毒試驗(yàn)中,免疫一次PLGA包裹的多價(jià)DNA疫苗產(chǎn)生了與免疫三次不加PLGA的多價(jià)DNA疫苗類似的保護(hù)效力。BCG疫苗加強(qiáng)策略在大動物牛中的實(shí)驗(yàn)表明,加強(qiáng)策略顯著提高了免疫牛中CD4+T細(xì)胞的比例,誘導(dǎo)了高水平的IFN-γ,且一直持續(xù)到攻毒后22周。 用結(jié)核桿菌強(qiáng)毒株感染小鼠,構(gòu)建潛伏期感染的動物模型。感染后8周,用異煙肼和吡嗪酰胺治療3個(gè)月。治療期間,用編碼三種結(jié)核桿菌抗原的基因聯(lián)合治療,使免疫應(yīng)答朝TH1型方向進(jìn)行,產(chǎn)生更多的TH1型的細(xì)胞因子如IFN-γ,使吞噬有結(jié)核桿菌的巨噬細(xì)胞從休眠狀態(tài)轉(zhuǎn)變?yōu)榛罨?以殺死結(jié)核桿菌,提高治療效率,降低載菌量,使治療時(shí)間減少一半,克服由于長期使用藥物產(chǎn)生耐藥性等問題。對開發(fā)有實(shí)際意義的治療用核酸疫苗具有重要的啟示。
[Abstract]:Tuberculosis and Brucellosis are zoonotic infectious diseases that seriously endanger human health and animal husbandry development. Vaccines are effective ways to control tuberculosis and brucellosis. Currently, BCG is the only vaccine to prevent tuberculosis, but its immune efficacy is unstable and unprotected in adults; S19 vaccine is used in bovine brucellosis, but not in adults. Therefore, efficient new vaccines are very important for controlling the spread of tuberculosis and brucellosis. Based on the study of tuberculosis and brucellosis vaccines, this paper focuses on the analysis of the mechanism of action of DNA vaccines, development status and improvement strategies.
Prokaryotic expression vectors encoding three antigens of Mycobacterium tuberculosis (Ag85B, MPT64 and MPT83) and three antigens of Brucella (BCSP31, SOD, and L7/L12) were constructed by molecular biology methods. The antigen proteins were induced to express and purified. The eukaryotic expression vectors of the six antigens were constructed for transient expression in vitro, coupled with GFP. Eukaryotic plasmids were extracted and prepared as DNA vaccines. After eight weeks of immunization, mice or cattle were infected with high doses of Mycobacterium tuberculosis or Brucella. The humoral immunity, cytokine levels, the number of cytokine-secreting cells and the activation ratio of T cells were studied. For example, the changes of T cell subtypes, the quantification of lethal T cells, and the amount of bacteria carried in the organs of infected animals were used to evaluate the advantages of each combination of vaccines and the immune responses induced by them, so as to interpret the efficacy of DNA vaccines and the mechanism of their adjuvants triggering immune responses. It can induce stronger TH1 immune response and enlarge the protective range. The protective efficacy is significantly higher than that of monovalent DNA vaccine.
By comparing the protective effects of the combined vaccine with different mineral adjuvants and gene adjuvants, it was confirmed that the adjuvant gene adjuvants IL-2, IL-12, IL-15, and the inorganic adjuvant DDA played an important role in inducing TH1 cell reaction and enhancing the function of killer T cells, especially the combination of six antigens DNA vaccine and IL-12. IL-15 adjuvant significantly increased the number of CD8+T cells secreting IFN-gamma. In vivo and in vitro experiments confirmed the important role of CD8+T cells in the process of combating Brucella. Polypeptide adjuvant KL5KLK increased APC cells. The ability of antigen uptake, processing and presentation prolongs the immune protection period, enhances the function of CD4 + T cells and the level of cell killing, which provides important evidence for clinical application.
The results showed that PLGA could significantly increase the expression of antigen and prolong the expression time. In the immune challenge test, the polyvalent DNA vaccine encapsulated by PLGA produced the same protection as the polyvalent DNA Vaccine Immunized with PLGA three times without PLGA. Protective effect. BCG vaccine reinforcement strategy in animal cattle showed that the reinforcement strategy significantly increased the proportion of CD4 + T cells in immunized cattle, induced high levels of IFN - gamma, and lasted until 22 weeks after infection.
Mice were infected with a virulent strain of Mycobacterium tuberculosis to establish an animal model of latent infection. Eight weeks after infection, the mice were treated with isoniazid and pyrazinamide for three months. Bacillus macrophages change from dormant state to active type in order to kill Mycobacterium tuberculosis, improve treatment efficiency, reduce bacterial load, reduce treatment time by half, overcome drug resistance and other problems caused by long-term use of drugs. It has important implications for the development of therapeutic nucleic acid vaccines with practical significance.
【學(xué)位授予單位】：北京大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392
本文編號：2242004
[Abstract]:Tuberculosis and Brucellosis are zoonotic infectious diseases that seriously endanger human health and animal husbandry development. Vaccines are effective ways to control tuberculosis and brucellosis. Currently, BCG is the only vaccine to prevent tuberculosis, but its immune efficacy is unstable and unprotected in adults; S19 vaccine is used in bovine brucellosis, but not in adults. Therefore, efficient new vaccines are very important for controlling the spread of tuberculosis and brucellosis. Based on the study of tuberculosis and brucellosis vaccines, this paper focuses on the analysis of the mechanism of action of DNA vaccines, development status and improvement strategies.
Prokaryotic expression vectors encoding three antigens of Mycobacterium tuberculosis (Ag85B, MPT64 and MPT83) and three antigens of Brucella (BCSP31, SOD, and L7/L12) were constructed by molecular biology methods. The antigen proteins were induced to express and purified. The eukaryotic expression vectors of the six antigens were constructed for transient expression in vitro, coupled with GFP. Eukaryotic plasmids were extracted and prepared as DNA vaccines. After eight weeks of immunization, mice or cattle were infected with high doses of Mycobacterium tuberculosis or Brucella. The humoral immunity, cytokine levels, the number of cytokine-secreting cells and the activation ratio of T cells were studied. For example, the changes of T cell subtypes, the quantification of lethal T cells, and the amount of bacteria carried in the organs of infected animals were used to evaluate the advantages of each combination of vaccines and the immune responses induced by them, so as to interpret the efficacy of DNA vaccines and the mechanism of their adjuvants triggering immune responses. It can induce stronger TH1 immune response and enlarge the protective range. The protective efficacy is significantly higher than that of monovalent DNA vaccine.
By comparing the protective effects of the combined vaccine with different mineral adjuvants and gene adjuvants, it was confirmed that the adjuvant gene adjuvants IL-2, IL-12, IL-15, and the inorganic adjuvant DDA played an important role in inducing TH1 cell reaction and enhancing the function of killer T cells, especially the combination of six antigens DNA vaccine and IL-12. IL-15 adjuvant significantly increased the number of CD8+T cells secreting IFN-gamma. In vivo and in vitro experiments confirmed the important role of CD8+T cells in the process of combating Brucella. Polypeptide adjuvant KL5KLK increased APC cells. The ability of antigen uptake, processing and presentation prolongs the immune protection period, enhances the function of CD4 + T cells and the level of cell killing, which provides important evidence for clinical application.
The results showed that PLGA could significantly increase the expression of antigen and prolong the expression time. In the immune challenge test, the polyvalent DNA vaccine encapsulated by PLGA produced the same protection as the polyvalent DNA Vaccine Immunized with PLGA three times without PLGA. Protective effect. BCG vaccine reinforcement strategy in animal cattle showed that the reinforcement strategy significantly increased the proportion of CD4 + T cells in immunized cattle, induced high levels of IFN - gamma, and lasted until 22 weeks after infection.
Mice were infected with a virulent strain of Mycobacterium tuberculosis to establish an animal model of latent infection. Eight weeks after infection, the mice were treated with isoniazid and pyrazinamide for three months. Bacillus macrophages change from dormant state to active type in order to kill Mycobacterium tuberculosis, improve treatment efficiency, reduce bacterial load, reduce treatment time by half, overcome drug resistance and other problems caused by long-term use of drugs. It has important implications for the development of therapeutic nucleic acid vaccines with practical significance.
【學(xué)位授予單位】：北京大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R392
【引證文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 藺國珍;布魯氏菌病LAMP檢測方法的建立及雙基因共表達(dá)分子疫苗研究[D];中國農(nóng)業(yè)科學(xué)院;2012年
，本文編號：2242004
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