| Lyme disease is a natural foci disease, which is caused by Borrelia burgdorferi sensu lato that mainly transmitted by ticks of Ixodes. Lyme disease is prevalent worldwide, and has become an serious vetor-borne disease exerting great threat to human health.(1) Totally 849 genomic DNA were extracted from seven tick species of five genus collected from eight provinces, and were used to screen Borrelia spp. based on fla gene by using nested-PCR. The results showed that Borrelia was detected in all of the provinces investigated, and the mean prevalence was 18.4%(156/849, 95% CI=12.8-42.5),ranging from 1.8%~77.4%. Sequence and phylogenetic analysis of obtained Fla genes indicated that four genotypies of Borrelia were identified, including B. garinii, B. burgdorferi sensu stricto, B. afzelii, B. americana as well as an unidentified species Borrelia sp. B. americana was first reported in China.(2) P66 is an outer membrane protein encoded by chromosome of Borrelia burgdorferi, with about 66 ku molecular weight. Current studies showed that this P66 protein primarily has two functions. First, as an adhension molecule, it can combine β3-integrin protein; Second, it plays an important role as a channel protein. In this study, the ORF of P66 gene was amplified by using primers designed based on the genomic data of B. garinii SZ and analysized by bioinformatic softwares. The results revealed that the ORF was 1866 bp, encoding 621 amino acid, with 68.4 ku molecular weight. Moreover, the signalpeptide of P66 protein located in 1-21 amino acid and α- and β-helix as the main secondary structure. Based on the phylogenetic analysis of P66 gene, B. garinii SZ was grouped with other B. garinii strains(USA-PBr strain and Germany-Pbi strain), indicating that P66 gene obtained was right.(3) The partial P66 gene was amplified using designed primers based on the above obtained P66 gene fragment. The partial fragment was linked into the pET-30a(+) vector, then transformed into Escherichia coli BL21(DE3) strain. After identified by PCR, double restriction enzyme digestion, and sequencing, the recombinant protein was expressed and purified. Polyclonal antibody was then prepared from New Zealand rabbit immunized with purified recombinant protein. Results showed that the recombinant protein was about 70 ku in size confirmed by SDS-PAGE, and Western-blot analysis indicated that the recombinant P66 protein could recognize the mouse monoclonal anti-His tag, positive sera of spirochete from mouse, and anti-P66 polyclonal antibody. Additionally, the anti-P66 polyclonal antibody could recognize native P66 protein.(4) The BALB/c mice were inoculated with B. garinii SZ through subcutaneous injection, and the different tissues of the infected mice were collected after infection in different times. Then, cDNA was synthesized based on the total RNA extracted from tissues, and relative real-time PCR was used to evaluate the expressive abundance of P66 in different tissues with flaB gene as the reference gene. The results showed that expression regularity of P66 gene was not obvious in tissues within different times, but the expression in brain changed significantly, indicating indirectly that P66 gene was relevant with the pathogenecity of B. garinii.These results demonstrated that P66 gene was related to the pathogenecity of B. garinii through bioinformatic analysis of P66, prokaryotic expression and polyclonal antibody preparation of P66 protein, and evalution for the expressive abundance of P66 in different tissues after infection by real-time PCR. |
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