發(fā)布時(shí)間：2018-02-01 01:18

  本文關(guān)鍵詞： 結(jié)核分枝桿菌 亞單位疫苗 Mtb10.4 HspX 多階段疫苗　出處：《蘭州大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：結(jié)核病由結(jié)核分枝桿菌感染引發(fā),是人類健康的一大威脅。在世界各地,隨著耐藥菌株和艾滋病病毒感染的增加,結(jié)核病仍具有較高的發(fā)病率和死亡率。結(jié)核分枝桿菌感染人體之后,可以以潛伏狀態(tài)存在很長(zhǎng)時(shí)間并且在人體免疫力低下時(shí)使被感染者發(fā)展為活動(dòng)性結(jié)核,因此潛伏期結(jié)核分枝桿菌的診斷和控制是人類面臨的一大難題。當(dāng)前結(jié)核病疫苗卡介苗(Bacilli Calmette Guerin, BCG),在全球很多地區(qū)得到廣泛應(yīng)用,但相關(guān)研究證實(shí)BCG可以預(yù)防兒童結(jié)核,但對(duì)成人肺結(jié)核和潛伏感染結(jié)核無(wú)效。因此,迫切需要研究針對(duì)成人結(jié)核病和潛伏感染結(jié)核病的新型疫苗和新型疫苗免疫策略。 目的：研究用于加強(qiáng)BCG效應(yīng)的有效結(jié)核亞單位疫苗,控制甚至根除包括持留菌在內(nèi)的各感染階段的結(jié)核分枝桿菌。本課題選用結(jié)核分枝桿菌對(duì)數(shù)生長(zhǎng)期抗原Mtb10.4(Rv0288)和休眠期保護(hù)性抗原HspX (Acr, Hsp16.3, Rv2031c),構(gòu)建無(wú)親和標(biāo)簽融合蛋白Mtb10.4-HspX(MH),并對(duì)其免疫特性和保護(hù)效果進(jìn)行評(píng)價(jià)。 方法：PCR擴(kuò)增基因Mtb10.4和HspX,并將他們依次插入到表達(dá)載體pET-30a(+)的多克隆位點(diǎn)中構(gòu)建重組質(zhì)粒Mtb10.4-HspX-pET30a(+),將此質(zhì)粒轉(zhuǎn)化入大腸桿菌BL-21(DE3)中表達(dá)融合蛋白MH。無(wú)標(biāo)簽融合蛋白MH通過(guò)三步連續(xù)層析法得到純化。用ELISPOT技術(shù),檢測(cè)MH對(duì)人外周血T細(xì)胞的免疫反應(yīng)。將該蛋白和佐劑二甲基三十六烷基銨(DDA)和海藻糖二霉菌酸脂(TDM)混合構(gòu)建亞單位疫苗。在0、3、6周,用MH疫苗連續(xù)免疫C57BL/6小鼠三次,設(shè)BCG和磷酸緩沖液(PBS)免疫組為對(duì)照,末次免疫后6周檢測(cè)此疫苗的免疫原性。MH疫苗在BCG初免后12、14周連續(xù)加強(qiáng)免疫C57BL/6小鼠兩次,BCG和PBS免疫組為對(duì)照,第20周(末次免疫后6周)檢測(cè)其加強(qiáng)BCG的免疫效果；第24周(末次免疫后10周)用H37Rv毒株經(jīng)尾靜脈注射攻擊被免小鼠,感染后42天,檢測(cè)小鼠肺臟組織中結(jié)核菌載量,并分析肺組織病理?yè)p傷程度,以此評(píng)價(jià)此疫苗加強(qiáng)BCG的保護(hù)效果。 結(jié)果：MH在大腸桿菌中可溶性穩(wěn)定表達(dá),且依次經(jīng)離子交換層析、疏水層析和凝膠過(guò)濾層析得到純化,純度達(dá)到95%；MH體外刺激結(jié)核病人及結(jié)核分枝桿菌潛伏感染人群的外周血淋巴細(xì)胞,可使其分泌大量干擾素γ(IFN-γ). MH亞單位疫苗單獨(dú)連續(xù)免疫小鼠三次,誘發(fā)小鼠產(chǎn)生大量抗原特異性細(xì)胞因子(IFN-γ、IL-17)和抗體(IgG1、IgG2b、IgG2c)。BCG初免MH加強(qiáng)組小鼠產(chǎn)生的細(xì)胞因子(IFN-γ、IL-17)和抗體(IgG1、IgG2b、IgG2c)水平明顯高于BCG單獨(dú)免疫組。毒株攻擊后,MH加強(qiáng)免疫組小鼠肺臟荷菌量明顯低于PBS組(P0.00)和BCG組(P=0.069),組織病理?yè)p傷較輕及且損傷面積明顯小于PBS(P0.05)和BCG(p0.05)組。 結(jié)論：MH對(duì)結(jié)核病患者、結(jié)核病潛伏感染者均顯示具有較強(qiáng)的免疫原性,聯(lián)合佐劑DDA-TDM免疫小鼠可引發(fā)較強(qiáng)的抗原特異性的細(xì)胞和體液免疫應(yīng)答且具有加強(qiáng)BCG的免疫保護(hù)效果。因此,MH聯(lián)合佐劑DDA-TDM有望成為針對(duì)活動(dòng)性結(jié)核病和潛伏感染感染結(jié)核病的有效候選疫苗。
[Abstract]:TB infection caused by Mycobacterium tuberculosis, is a major threat to human health. In the world, with the increase in resistant strains and HIV infection, tuberculosis still has high morbidity and mortality. After human infection with Mycobacterium tuberculosis, latent state can exist for a long time and make the infected development activities tuberculosis in immunocompromised, so the diagnosis and control of latent Mycobacterium tuberculosis is a major problem facing mankind. The TB vaccine (Bacilli Calmette, Guerin, BCG) is widely used in many areas of the world, but the research confirmed that BCG can prevent children from tuberculosis, but of adult pulmonary tuberculosis and latent infection tuberculosis is invalid. Therefore, it is urgent to study the new vaccine for adult tuberculosis and latent infection of tuberculosis and new vaccine strategies.
Objective: To study the effective for tuberculosis subunit vaccine BCG strengthening effect, control and eradicate Mycobacterium tuberculosis including persisters, each stage of infection. This topic selection of Mycobacterium tuberculosis antigen Mtb10.4 in logarithmic growth phase (Rv0288) and dormant protective antigen HspX (Acr, Hsp16.3, Rv2031c), construction of affinity tags the fusion protein Mtb10.4-HspX (MH), and to evaluate its immunogenicity and protective effect.
Methods: PCR gene amplification of Mtb10.4 and HspX, and they were inserted into the expression vector pET-30a (+) to construct the recombinant plasmid of Mtb10.4-HspX-pET30a multiple cloning sites (+). The plasmid was transformed into E.coli BL-21 (DE3) in the expression of fusion protein MH. tag fusion protein MH was purified by three steps of continuous chromatography. ELISPOT, MH immunoreactivity was detected on human peripheral blood T cells. The protein and adjuvant two methyl thirty-six alkyl ammonium (DDA) and trehalose two mycolates (TDM) hybrid construct subunit vaccine. In 0,3,6 weeks, C57BL/6 mice were immunized with MH vaccine for three times, BCG and phosphate buffer (PBS) immune group, 6 weeks after the final immunization was detected by the vaccine immunogenicity of.MH vaccine in BCG after immunization of 12,14 weeks to strengthen the immune C57BL/6 mice two times, BCG and PBS immune group, Twentieth weeks (6 weeks after the last immunization and its detection) The immune effect of BCG was strong. After twenty-fourth weeks (10 weeks after the last immunization), the mice were attacked by the tail vein injection of H37Rv strain. After 42 days of infection, the amount of Mycobacterium tuberculosis in lung tissue of mice was detected, and the pathological degree of lung tissue was analyzed, so as to evaluate the protective effect of the vaccine on BCG.
Results: MH in Escherichia coli and stable expression, sequentially through ion exchange chromatography, hydrophobic chromatography and gel filtration chromatography purified, the purity reached 95%; peripheral blood lymphocytes stimulated with MH in vitro, tuberculosis and latent Mycobacterium tuberculosis infection, the secretion of interferon gamma (IFN- gamma) MH subunit. Continuous vaccine alone immunized mice three times, induced by a large number of antigen specific cytokine (IFN-, IL-17) and antibodies (IgG1, IgG2b, IgG2c) cytokine free MH mice produced at the beginning of.BCG strengthening (IFN-, IL-17) and antibodies (IgG1, IgG2b, IgG2c) was significantly higher than that of BCG alone immune group. After the attack of MH strains, strengthen the bacterial load of immunized mice lung was significantly lower than in group PBS (P0.00) and BCG group (P=0.069), pathological damage and lighter and damage area was less than that of PBS (P0.05) and BCG (P0.05) group.
緇撹錛歁H瀵圭粨鏍哥梾鎮(zhèn)ｈ，

本文編號(hào)：1480517