| Objective:1. To quantify the burden of Borrelia burgdorferi in different representative tissues in the murine host.2. To identify the joint-specific expression profile of Borrelia burgdorfri in the murine hosts.3. To clone and highly express Borrelia recombinant bmpA, Genomic DNA of Borrelia burgdorferi reference strain B31is as template, using PCR to amplify bmpA gene sequences, directional cloning and high expression and purification of recombinant bmpA. To identify the subcellular location of bmpA.4. To establish Lyme arthritis model of KM mouse by injecting recombinant Borrelia burgdoferi membrance protein A(rBmpA) regularly and locally to the tibial tarsal joint and investigate the arthritic changes in X-ray and histopathological levels, on this basis, detect the contents of Th-17cell-associated IL-6, TGF-β and IL-17cytokines in the serum and synovial fluid of Lyme arthritis model of KM mouse and explore the role of Th-17cells in the pathogenesis of Lyme arthritis.5. To explore the roles of BmpA to activate the macrophages collected from peritoneal cavity, and murine lymphocytes from spleen, determine the effects of BmpA on the activation of macrophages and lymphocytes.Methods:1. To cultivate the low-passage Borrelia burgdorferi to logarithmic growth phase, and dilute bacterial culture to a concentration of1×105/ml for experimental use. To establish a Borrelia burgdorferi-infected murine model, mice were injected100μl diluted culture intradermally in the back. After artificial infection was confirmed, mice were sacrificed at day12and18postinfection in CO2box,skin, joints heart, and urine bladder samples were collected aseptically and frozen at-80℃, total DNA extracted, Borrelia burgdorferi flaB was quantified by real time quantitative PCR(Q-PCR). Data were analyzed statistically to determine if bacterial burdens in various tissues show a significant difference.2. Establishing the murine model of Lyme disease, sacrificing the infected mice at different time-points, collecting the joint, heart, skin, and urinary bladder samples of the infected mice, and extracting total RNA from the samples. DECAL technique and microarray were applied to btain the transcriptomes of Borrelia burgdorferi in the joints, heart, skin, and urinary bladder, the transcriptomes from different tissues were compared to identify the joint-specific expression profile of Borrelia burgdorferi.3.(1) BmpA gene cloning and the production of recombinant protein.Genomic DNA of Borrelia burgdorferi reference strain B31is as template, Designing primers, using PCR to amplify bmpA genome sequences, BmpA gene was cloned into the expression vector pGEX-6p-1, restriction enzyme digestion, transformed into E.coli strain BL21, get bmpA recombinant strain.(2) High level expression and purification of recombinant bmpA:From recombinant bacteria culture temperature, induction time, inducer dose, OD600optimal inducing conditions, etc, finding the best solution of High Expression Recombinant bmpA, Purified by GSH re bmpA, to explore the optimal conditions purified bmpA.(3) Identify the subcellular location of BmpA.4.(1) To validate the role of rBmpA in the induction of Lyme arthritis: Optimize the concentration, dose, frequency of rBmpA injected to verify the role of rBmpA in the induction of Lyme arthritis.(2) To establish animal models of Lyme arthritis in the new animal strains (KM mouse) and in the use of the new method (direct articular injection):Explore the new way of taking local tibial tarsal joint injection of KM mouse to establish the model of Lyme arthritis.(3)To explore the role of IL-6, IL-17, TGF-β-associated Th-17cells in the pathogenesis of Lyme arthritis. To detect the contents of IL-6, IL-17, TGF-β in the serum and synovial fluid of Lyme arthritis model of KM mouse to explore the role of Th-17cells in the pathogenesis of Lyme arthritis.5. To use BmpA to activate the macrophages collected from peritoneal cavity, and murine lymphocytes from spleen, explore the effects of BmpA on the activation of macrophages and lymphocytes.Result:1. Spirochete burden is highest in the urine bladder, medium in the skin and joint, lowest in the heart.2. Borrelia burgdorferi expresses21joint-specific genes, including13genes located in chromosome and9genes located in plasmids at day15after infection, and expresses24genes, including13genes in chromosome and11genes in plasmids at day105after infection. Borrelia burgdorferi shows specific gene expression profile in the murine joints. The sprochetal genes which express most highly in murine joints are bmpA and bmpB.3.(1) Target bands and peaks were appeared on level of gene and protein, showing the expression vector bmpA-pGEX-6p-1was successfully constructed and expressed recombinant bmpA.(2) through analysis and comparision, maximal expression of recombinant plasmid was induced by O.lmmol/ml IPTG at37℃for6h when OD value of bacterium was0.5-1.0.(3) the optimal expression conditions of recombinant1L can be purified to2.9-3.1mg of bmpA protein.(4) BmpA is located on the surface of Borrelia burgdorferi outer member.4.(1) rBmpA is closely related to the pathogenesis of Lyme Arthritis.(2)Successfully established Lyme arthritis model of KM mouse by using the way that directly injects rBmpA into tibial tarsal joint of KM mice locally and found the new way of establishing Lyme Arthritis animal model.(3) Detectd the contents of Th-17cell-associated IL-6, IL-17, TGF-β in the serum and synovial fluid of Lyme Arthritis model of KM mouse and compared it with those of the negative control group and normal group, results have no statistical difference.Finally, the relevant reasons were discussed and suggestions for further research were promoted. 5. Our preliminary results showed that Lyme spirochete BmpA can hardly activate the macrophages collected from peritoneal cavity, but can strongly activate murine lymphocytes from spleen.Conclusion:1. Difference of spirochete burden in various tissues is significant. Spirochete burden in the tissue is not positive related to the severity of this tissue.2. Borrelia burgdorferi expresses some specific genes different from those in the other tissues in infected murine joints. These joint-specific genes might be involved in the survival and arthritis pathogenesis of Borrelia burgdorferi.3.(1) As effort of our lab, prokaryotic expression system of E.coli of was successfully constructed, it was identified on gene and protein level.(2) A high expression of recombinant bmpA was accomplished, With purified recombinant GST bmpA, to explore the optimal conditions for purification of recombinant bmpA.(3) bmpA is located in the spirochetal cell surface.4.(1) Successfully established Lyme arthritis model of KM mouse by using the way that injecting rBmpA diluent into tibial tarsal joint of KM mice locally.(2) rBmpA is closely related with the attack of Lyme Arthritis.5. Our preliminary results showed that Lyme spirochete BmpA can hardly activate the macrophages collected from peritoneal cavity, but can strongly activate murine lymphocytes from spleen. |
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