结核分枝杆菌亚单位疫苗Mtb10.4-HspX的构建及效果评价
发布时间：2018-02-01 01:18
本文关键词： 结核分枝杆菌 亚单位疫苗 Mtb10.4 HspX 多阶段疫苗 出处：《兰州大学》2012年硕士论文 论文类型：学位论文
【摘要】：结核病由结核分枝杆菌感染引发,是人类健康的一大威胁。在世界各地,随着耐药菌株和艾滋病病毒感染的增加,结核病仍具有较高的发病率和死亡率。结核分枝杆菌感染人体之后,可以以潜伏状态存在很长时间并且在人体免疫力低下时使被感染者发展为活动性结核,因此潜伏期结核分枝杆菌的诊断和控制是人类面临的一大难题。当前结核病疫苗卡介苗(Bacilli Calmette Guerin, BCG),在全球很多地区得到广泛应用,但相关研究证实BCG可以预防儿童结核,但对成人肺结核和潜伏感染结核无效。因此,迫切需要研究针对成人结核病和潜伏感染结核病的新型疫苗和新型疫苗免疫策略。 目的：研究用于加强BCG效应的有效结核亚单位疫苗,控制甚至根除包括持留菌在内的各感染阶段的结核分枝杆菌。本课题选用结核分枝杆菌对数生长期抗原Mtb10.4(Rv0288)和休眠期保护性抗原HspX (Acr, Hsp16.3, Rv2031c),构建无亲和标签融合蛋白Mtb10.4-HspX(MH),并对其免疫特性和保护效果进行评价。 方法：PCR扩增基因Mtb10.4和HspX,并将他们依次插入到表达载体pET-30a(+)的多克隆位点中构建重组质粒Mtb10.4-HspX-pET30a(+),将此质粒转化入大肠杆菌BL-21(DE3)中表达融合蛋白MH。无标签融合蛋白MH通过三步连续层析法得到纯化。用ELISPOT技术,检测MH对人外周血T细胞的免疫反应。将该蛋白和佐剂二甲基三十六烷基铵(DDA)和海藻糖二霉菌酸脂(TDM)混合构建亚单位疫苗。在0、3、6周,用MH疫苗连续免疫C57BL/6小鼠三次,设BCG和磷酸缓冲液(PBS)免疫组为对照,末次免疫后6周检测此疫苗的免疫原性。MH疫苗在BCG初免后12、14周连续加强免疫C57BL/6小鼠两次,BCG和PBS免疫组为对照,第20周(末次免疫后6周)检测其加强BCG的免疫效果；第24周(末次免疫后10周)用H37Rv毒株经尾静脉注射攻击被免小鼠,感染后42天,检测小鼠肺脏组织中结核菌载量,并分析肺组织病理损伤程度,以此评价此疫苗加强BCG的保护效果。 结果：MH在大肠杆菌中可溶性稳定表达,且依次经离子交换层析、疏水层析和凝胶过滤层析得到纯化,纯度达到95%；MH体外刺激结核病人及结核分枝杆菌潜伏感染人群的外周血淋巴细胞,可使其分泌大量干扰素γ(IFN-γ). MH亚单位疫苗单独连续免疫小鼠三次,诱发小鼠产生大量抗原特异性细胞因子(IFN-γ、IL-17)和抗体(IgG1、IgG2b、IgG2c)。BCG初免MH加强组小鼠产生的细胞因子(IFN-γ、IL-17)和抗体(IgG1、IgG2b、IgG2c)水平明显高于BCG单独免疫组。毒株攻击后,MH加强免疫组小鼠肺脏荷菌量明显低于PBS组(P0.00)和BCG组(P=0.069),组织病理损伤较轻及且损伤面积明显小于PBS(P0.05)和BCG(p0.05)组。 结论：MH对结核病患者、结核病潜伏感染者均显示具有较强的免疫原性,联合佐剂DDA-TDM免疫小鼠可引发较强的抗原特异性的细胞和体液免疫应答且具有加强BCG的免疫保护效果。因此,MH联合佐剂DDA-TDM有望成为针对活动性结核病和潜伏感染感染结核病的有效候选疫苗。
[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.
缁撹锛歁H瀵圭粨鏍哥梾鎮ｈ，
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