Borrelia Burgdorferi Advantage The Antigen Bmpa Molecular Cloning, Predominantly Expressed And Purified

Objective:1. Genomic DNA of Borrelia burgdorferi reference strain B31 was as template, using PCR to amplify bmpA gene sequences, directional cloning and high expression and purification of recombinant BmpA.2. The sensitivity and specificity of rBmpA was assessed by ELISA in the serological diagnosis of Lyme disease.Methods:1. bmpA gene cloning and the production of recombinant proteinGenomic DNA of Borrelia burgdorferi reference strain B31 was 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 rBmpA:From recombinant bacteria culture temperature, induction time, inducer dose, OD600 optimal inducing conditions, etc, finding the best solution of high expression rBmpA, purified by GSH, and explored the optimal conditions to purify rBmpA3. rBmpA the diagnosis of Lyme disease in the initial applicationWith Borrelia burgdorferi infection in serum and in healthy volunteers, the sensitivity and specificity of rBmpA was assessed by ELISA in the serological diagnosis of Lyme disease.Result:1. Target bands and peaks were appeared on level of gene and protein,determined the expression vector bmpA-pGEX-6p-1 was successfully constructed and expressed rBmpA.2. Through analysis and comparision, maximal expression of recombinant plasmid was induced by 0.1mmol/ml IPTG at 37℃for 6h when OD value of bacterium was 0.5-1.0.3. The optimal expression conditions of recombinant 1L can be purified to 2.9-3.lmg of BmpAprotein.Conclusion: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. Determine a high expression of recombinant BmpA the best solution, With purified recombinant GST-BmpA, to explore the optimal conditions for purification of rBmpA3. In the serological diagnosis of Lyme disease the sensitivity of rBmpA was high but specificity of rBmpA was low.