Isolation And Identification Of Borrelia Burgdorferi Form Tick And ELISA Method For Lyme Disease Research

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.