【作者】 仝彩玲；

【导师】 李培英； 周金林；

【作者基本信息】 安徽农业大学 ， 临床兽医学， 2010， 硕士

【摘要】 莱姆病是经硬蜱传播的,由伯氏疏螺旋体引起的人畜共患病。该病呈世界性分布,目前,全球估计年发病人数约为30万人左右,且有不断增长的趋势。在美国,莱姆病有“第二艾滋病”之称,1992年世界卫生组织将此病列为重点防治对象,现已成为全球性的公共卫生问题之一。近年来,对莱姆病病原生物学、致病机理、流行病学、诊断及综合防治方面的研究已取得了一定的成果,但由于伯氏疏螺旋体抗原结构的复杂性以及莱姆病的临床症状的多样性,使得该病的确诊较困难。另外,莱姆病作为一种新发病,在我国对其传播媒介蜱的种类尚未完全明晰,同时关于其病原体的基因型及分类地位均未阐明,这给诊断和防治该病带来极大的挑战。因此,阐明其传播媒介的种类和病原体的分类地位将有助于我们建立高效、快速诊断手段,合理地开展该病的防治工作。本文主要在莱姆病病原体的分类学及其诊断学两个方面进行探索。(1)对源于我国部分林区采集的亚洲璃眼蜱(Hyalomma asiaticum)、长角血蜱(Haemaphysalis longicornis)等多种蜱种进行伯氏疏螺旋体的检测;(2)对所分离的螺旋体进行分型研究;(3)对所获得的伯氏疏螺旋体鞭毛基因及鞭毛基因的保守区段进行克隆,并在体外表出达伯氏疏螺旋体鞭毛基因保守区段,用保守区段重组蛋白作为莱姆病的诊断抗原,建立莱姆病ELISA诊断方法。第一部分,用套式PCR方法扩增蜱体内的伯氏疏螺旋体鞭毛基因,对采自新疆、青岛和延边等地区的亚洲璃眼蜱和长角血蜱及蜱肠培养物进行检测,对PCR所扩增目的基因片段进行测序,测序结果经GenBank中的blast比对分析。采用BSK-H固体培养基分离培养技术,从蜱肠内分离和纯化伯氏疏螺旋体单克隆,并采用分子生物学方法对分离到的单克隆菌落进行分型。首先对新疆地区的亚洲璃眼蜱和镰形扇头蜱在显微镜下进行解剖,取其肠,用BSK-H液体培养基进行培养,收集蜱肠培养物,提取其基因组DNA,采用套式PCR方法扩增鞭毛基因。然后,对经鉴定含有伯氏螺旋体的肠培养物,利用BSK-H固体培养分离技术,对其进行分离纯化,建立了一种快速而方便的分离伯氏疏螺旋体培养方法。最后,应用recA基因和5S-23S间区基因作为分型的靶基因对分离到的伯氏疏螺旋体单克隆进行初步分型,recA基因是与伯氏疏螺旋体进化相关的基因,其GC含量的变化可以通过荧光定量PCR的熔解曲线很容易的反映出来,根据此特性,用定量荧光PCR熔解曲线对分离到的螺旋体单克隆进行初步分型。经初步分型,本次从亚洲璃眼蜱中分离到伽氏疏螺旋体、狭义的伯氏疏螺旋体等菌株。同时为进一步确定螺旋体基因型,对其5S-23S间区进行PCR扩增,并对扩增产物进行测序,根据其测序结果进一步分型。伯氏疏螺旋体单克隆的分离及初步分型的研究,为莱姆病病原生物学研究奠定了基础。第二部分,对莱姆病血清学诊断技术进行了研究。该研究用重组伯氏疏螺旋体鞭毛基因蛋白作为诊断抗原,建立莱姆病ELISA诊断方法。本研究利用生物工程方法,选取鞭毛基因的氨基酸序列的第131-266区段作为目的片段,对其克隆,连接到PGEX-4T-1表达载体上,构建保守基因片段的重组质粒,然后转化到感受态细胞BL21中,原核表达伯氏疏螺旋体鞭毛基因抗原性和保守性较强的区段的平截性蛋白,用GST.BIND纯化系统纯化伯氏疏螺旋体鞭毛基因重组蛋白,所纯化的蛋白用作诊断抗原;同时将伯氏疏螺旋体分别接种小鼠和兔,两周后收集血清,用上述诊断抗原鉴定,发现经感染的小鼠和兔血清均为阳性血清,建立了敏感性和特异性较高的ELISA诊断方法。此方法为解决莱姆病诊断困难、难以确诊提供了一种有益的借鉴,特别为莱姆病早期的诊断提供了一种有力的手段,且有一定的临床应用价值。综上所述,本研究用简便快捷的固体培养方法对伯氏疏螺旋体单克隆分离株进行分离,并筛选出一种对我国莱姆病病原体分型新方法;建立了检测特异性强、敏感性高的莱姆病血清抗体的ELISA方法。这为有效、快速诊断以及合理防治莱姆病提供理论依据,同时也为该病的流行病学的调查工作开展提供了有力的手段。更多还原

【Abstract】 Lyme disease is a zoonosis which is caused by Borrelia burgdorferi and transmitted via tick, and it can cause many organs and systems injured. Lyme disease is widely distributed in the word. Nowadays, there are about 0.3 million cases of lyme disease were reported every year, but this tendency is increasing. It is called“the second AIDS”in American. In 1992, it is listed as the main prevent disease by the World Health Organization, nowadays, it become one of the global health problem. Recently, great progreses have been made in many parts of Lyme disease, including pathogen, pathogenesis, epidemiolog, diagnosis and integrated control, but Lyme disease in diagnosis is difficult because of the complex antigenic structure and multiplicity clinical symptoms. Otherwise, as a new disease, the sort and distribution of tick, which can tansmitted it, still clearly got elucidation in China, meanwhile the genetypes of its pathogen are uninterrptly recorded, which will take challege for preventing and controling it. Therefore, to elucidate the sorts of intermediary ticks and classifying of the pathogen, it will be helpful for its rapid diagnosis and controling.In the thesis, it composed of two parts involving in the etiological agent characterization and its diagnosis. Firstly, we detect Borrelia burgdorferi from Hyalomma asiaticum ,Haemaphysalis longicornis and its’eggs originated from Xinjiang, Jilin, and Shandong forest. Secondly, the typing study of isolated helicoid was made. Thirdly, flagellin gene of Borrelia burgdorferi and its conservative segment were cloned respectively, and the conservative segment was expressed in vitro to establish the ELISA for its diagnosis.In the part one, nested PCR was applied to determine the flagellin gene of Lyme burgdorferi to find the carrier with borrelia burgdorferi of tick. Borrelia burgdorferi were isolated from Hyalomma asiaticum and Haemaphysalis longicornis, originated from Xinjiang, Jilin, and Shandong forest, genemic DNA was extrated, the aim gene was amplified, sequenced, and analysed by blast. Then, Borrelia burgdorferi from ticks was isolated and cloned by the BSK solid culture technology, and its typing was made by molecular biology. Firstly, the ticks were dissected under microscope to obtain their guts, and the guts were cultured in BSK-H liquid medium, then the cultures were collected and their genemic DNA was extracted, the nested PCR was applied to amplify the flagellin gene of Borrelia burgdorferi. BSK-H solid medium was used to isolate the clone of borrelia burgdorferi from the positive cultures which were identified by PCR.The reslut indicated that a rapid and convenient separation of Borrelia burgdorferi was established. Finally, initial typing of Borrelia burgdorferi was made by application of recA genes and the 5S-23S inter-region genes. recA gene is related with Borrelia burgdorferi in evolutionthe, and its GC content changes can be determined by fluorescence quantitative PCR curves easily. Based on this feature, we took quantitative fluorescent PCR to type it initially. After initial classification, Borrelia garinii, Borrelia burgdorferi sensu stricto and others from Hyalomma asiaticum. In order to make further determination of the type of spirochete, PCR was amplified 5S-23S inter-region, the amplified product was sequenced and analysed. The study of isolation of Borrelia burgdorferi and typing could be play foundation for pathogenic biology of Lyme disease.In the part three, the study of diagnostic technique of serology in Lyme disease was made, and the ELISA was established with the recombinant protein. The aim fragment of 131-266 region of flagellar gene was cloned into vector of PGEX-4T-1 by bio-engineering, transformed into E.ciol BL21 competent cells, and expressed. The expressed protein was puried with the purification system of GST.BIND and used for diagnosis. Meanwhile, mice and rabbit were injected with Borrelia burgdorferi to prepare for positve serum, the ELISA with high sensitivity and specificity was set up. It settles for the difficulty in diagnosis to Lyme disease, and provide for the potent measure to diagnose it in clinic.To sum up, Monoclone of Lyme disease spirochetes was isolated with convenient and shortcut solid culture, and the new type mean of isolation was ensured; the ELISA with sensitivity and specifity for detecteting the disease was established. The conclusion could be made that it provides the reference for its diagnosis and controling and the mean for the investigation of its epidemiology.更多还原