Molecular Cloning, Sequence Analysis Of 5s-23srRNA Intergenic Spacer DNA And OSPA Expression Of Four Chinese Borrelia Burgdorferi

Objective: To investigate the genetic characterization and the variation of OSPA gene in four Chinese isolates of Borrelia burgdorferi; and express outer surface protein A(OSPA) in vitro. Methods: PCR technique was used to amplify the 5s-23srRNA intergenic spacer DNA and OSPA gene from whole cellular DNA of GXLD-4,9,18 and Chang 14. The amplified products were cloned into plasmid pGEM-T Easy and sequenced. The four recombinant plasmids pGEM-T Easy-OSPA were cleavaged by Ndel and Xhol restriction enzymes and cloned into an expression vector PET30a respectively. Four recombinant plasmids pET30a-OSPA expressed in E.coii BL21. Results: The 5s-23srRNA intergenic spacer DNA of GXLD-4,9,18 and Chang 14 were 242bp, revealed a nucleotide sequence homology of>99%. the four isolates had higher sequence homology with Borrelia valaisiana than with other genospecies .The OSPA genes amplified from four isolates were 774bp and encode 258 amino acids. The homogeneity of nucleotides and amino acids sequence among the four strains was 98.7-99.8% and 98-99% respectively. It had 99.8% nucleotides and 99%amino acids sequence homology between GXLD-4 and Chang 14. The four isolates had higher sequence homology with 10MT and Ya501. OSPA proteins were expressed heavily. Conclusion: The four isolates belong to Borrelia valaisiana genospecies and had the similar OSPA serotyping with 10MT, Ya501.