问号状赖型钩端螺旋体LAg42膜蛋白基因的克隆表达及功能研究

发布时间：2018-10-23 11:24

【摘要】： 钩端螺旋体病是由致病性钩端螺旋体感染引起的一种人兽共患病，它在世界范围内广泛流行，严重威胁着人类的健康。我国是钩体病疫情较为严重的国家，钩体病的防治是一项重要的任务。目前，在钩体病的治疗上已取得重大进展，但对其致病机制及免疫分子机理的认识还不甚清楚，现有的预防性疫苗亦仅能产生短期的、不完全的免疫保护效应。钩体菌型复杂，仅致病性钩体就有超过230种血清型，这种血清型的多样性主要是由于LPS碳水化合物成分的差异性引起的。虽然钩体LPS有一定的免疫保护性，但不同的血清型具有不同的LPS成分，所以以LPS成分研制的疫苗难以产生完全性保护作用。然而研究表明，致病性钩体蛋白提取物可以对多种血清型钩体的感染产生广泛的交叉保护作用，是潜在的侯选疫苗，如LipL32(也被称作Hap1)，LipL41和OmpL1已经被发现能产生保护性效应，证明它们是各种致病性钩体中的保护蛋白。 最近，Nobuo Koizumi等用全钩抗血清结合Western blotting的方法在L.manilae UP-MMC菌株基因组中发现了一种新的蛋白抗原LAg42，并预测它可能是钩端螺旋体的毒力因子，可以引起保护性免疫反应，但尚未对其进行实验研究。我们以赖型钩体中编码LAg42膜蛋白的基因为对象，对其进行克隆分析，进行原核及真核表达，及LAg42膜蛋白的功能研究。 本研究首先以问号状赖型钩体017株、56601株及双曲钩体PatocⅠ株基因组为模板，以PCR扩增目的基因，构建Lag42基因与质粒载体pET-32a(+)的原核重组表达质粒pET-lag42，克隆筛选并测序；并利用Dnaman、NCBI及SignalP等生物信息资源对其进行序列分析；最后对其进行原核表达，分离纯化融合蛋白后制备多价抗体，并用XTT法和流式细胞仪对纯化的LAg42膜蛋白进行初步的功能研究。同时，构建Lag42与pcDNA3.1A~+的真核重组表达质粒pcDNA3.1A~+-lag42；并利用脂质体介导pcDNA3.1-Loa22转染COS7细胞，RT-PCR检测其表达情况。 研究结果显示不同毒力赖型钩体均能扩增出目的片段，而PatocⅠ株则未能扩增出目的片段。序列分析显示不同赖型钩体Lag42基因的同源性很高，pET-lag42转化入大肠杆菌BL21后，表达出分子量约为62kDa的LAg42融合蛋白，在适当的条件下，融合蛋白在菌体内主要以可溶性方式(非包涵体)表达，经亲和层析纯化，获得高纯度的LAg42融合蛋白，，XTT和流式细胞仪检测结果显示出纯化LAg42融合蛋白的细胞毒性。RT-PCR检测显示pcDNA3.1A~+-lag42能在哺乳动物细胞中表达。 综上所述，本研究对LAg42膜蛋白基因进行了克隆分析，成功地进行了原核及真核表达，并对LAg42膜蛋白的功能进行了初步的实验研究，为钩体新型疫苗的深入研究提供了新的实验资料。
[Abstract]:Leptospirosis is a zoonosis caused by pathogenic leptospirosis. Leptospirosis is a serious epidemic situation in China, and the prevention and control of leptospirosis is an important task. At present, great progress has been made in the treatment of leptospirosis, but the understanding of its pathogenesis and immune molecular mechanism is not clear, and the existing prophylactic vaccine can only produce short-term, incomplete immune protective effect. There are more than 230 serotypes in the pathogenic leptospira alone. The diversity of these serotypes is mainly due to the difference of carbohydrate composition of LPS. Although Leptospira LPS has a certain immune protection, different serotypes have different LPS components, so it is difficult to develop a complete protective effect of Leptospira LPS vaccine. However, studies have shown that the pathogenic leptospirosis protein extract has a wide cross-protection against infection of many serotypes of leptospirosis and is a potential candidate vaccine, such as LipL32 (also known as Hap1), LipL41 and OmpL1 have been found to have protective effects. They are proved to be protective proteins in various pathogenic leptospirosis. Recently, Nobuo Koizumi and others have found a new protein antigen LAg42, in the genome of L.manilae UP-MMC strain by the method of whole hook antiserum binding Western blotting and predicted that it may be a virulence factor of leptospira, which can induce protective immune response. But no experimental study has been carried out on it. The gene encoding LAg42 membrane protein in Leptospira lanceolaris was cloned and analyzed for prokaryotic and eukaryotic expression, and the function of LAg42 membrane protein was studied. In this study, the genomes of 56601 Leptospira interrogans, 56601 Leptospira and Patoc 鈪

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