【摘要】： 鉤端螺旋體病是由致病性鉤端螺旋體感染引起的一種人獸共患病，它在世界范圍內(nèi)廣泛流行，嚴(yán)重威脅著人類的健康。我國是鉤體病疫情較為嚴(yán)重的國家，鉤體病的防治是一項重要的任務(wù)。目前，在鉤體病的治療上已取得重大進(jìn)展，但對其致病機制及免疫分子機理的認(rèn)識還不甚清楚，現(xiàn)有的預(yù)防性疫苗亦僅能產(chǎn)生短期的、不完全的免疫保護(hù)效應(yīng)。鉤體菌型復(fù)雜，僅致病性鉤體就有超過230種血清型，這種血清型的多樣性主要是由于LPS碳水化合物成分的差異性引起的。雖然鉤體LPS有一定的免疫保護(hù)性，但不同的血清型具有不同的LPS成分，所以以LPS成分研制的疫苗難以產(chǎn)生完全性保護(hù)作用。然而研究表明，致病性鉤體蛋白提取物可以對多種血清型鉤體的感染產(chǎn)生廣泛的交叉保護(hù)作用，是潛在的侯選疫苗，如LipL32(也被稱作Hap1)，LipL41和OmpL1已經(jīng)被發(fā)現(xiàn)能產(chǎn)生保護(hù)性效應(yīng)，證明它們是各種致病性鉤體中的保護(hù)蛋白。 最近，Nobuo Koizumi等用全鉤抗血清結(jié)合Western blotting的方法在L.manilae UP-MMC菌株基因組中發(fā)現(xiàn)了一種新的蛋白抗原LAg42，并預(yù)測它可能是鉤端螺旋體的毒力因子，可以引起保護(hù)性免疫反應(yīng)，但尚未對其進(jìn)行實驗研究。我們以賴型鉤體中編碼LAg42膜蛋白的基因為對象，對其進(jìn)行克隆分析，進(jìn)行原核及真核表達(dá)，及LAg42膜蛋白的功能研究。 本研究首先以問號狀賴型鉤體017株、56601株及雙曲鉤體PatocⅠ株基因組為模板，以PCR擴增目的基因，構(gòu)建Lag42基因與質(zhì)粒載體pET-32a(+)的原核重組表達(dá)質(zhì)粒pET-lag42，克隆篩選并測序；并利用Dnaman、NCBI及SignalP等生物信息資源對其進(jìn)行序列分析；最后對其進(jìn)行原核表達(dá)，分離純化融合蛋白后制備多價抗體，并用XTT法和流式細(xì)胞儀對純化的LAg42膜蛋白進(jìn)行初步的功能研究。同時，構(gòu)建Lag42與pcDNA3.1A~+的真核重組表達(dá)質(zhì)粒pcDNA3.1A~+-lag42；并利用脂質(zhì)體介導(dǎo)pcDNA3.1-Loa22轉(zhuǎn)染COS7細(xì)胞，RT-PCR檢測其表達(dá)情況。 研究結(jié)果顯示不同毒力賴型鉤體均能擴增出目的片段，而PatocⅠ株則未能擴增出目的片段。序列分析顯示不同賴型鉤體Lag42基因的同源性很高，pET-lag42轉(zhuǎn)化入大腸桿菌BL21后，表達(dá)出分子量約為62kDa的LAg42融合蛋白，在適當(dāng)?shù)臈l件下，融合蛋白在菌體內(nèi)主要以可溶性方式(非包涵體)表達(dá)，經(jīng)親和層析純化，獲得高純度的LAg42融合蛋白，，XTT和流式細(xì)胞儀檢測結(jié)果顯示出純化LAg42融合蛋白的細(xì)胞毒性。RT-PCR檢測顯示pcDNA3.1A~+-lag42能在哺乳動物細(xì)胞中表達(dá)。 綜上所述，本研究對LAg42膜蛋白基因進(jìn)行了克隆分析，成功地進(jìn)行了原核及真核表達(dá)，并對LAg42膜蛋白的功能進(jìn)行了初步的實驗研究，為鉤體新型疫苗的深入研究提供了新的實驗資料。
[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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