Establishment Of PCR-DHPLC Fingerprints For Identifying Lyme Disease Spirochetes And Fusion Expression And Purification Of Borrelia Burgdorferi OspA

Objective:In our study,Borrelia burgdorferi genes were accurately, rapidly, and high-fluxdetected by application of PCR–DHPLC method,and prokaryotic expression of OutSurface ProteinA (OspA) provided a basis for the subsequent colloidal gold stripdetection of Lyme disease spirochetes.Methods:With worldwide there are three common kinds Lyme disease spirochetespathogenic strains:B.buredorferi sensu stricto,B.garinii,B.afzelii as the researchobject. OspC as species-specific gene sequence was selected to design universalprimers by Genbank and application of PCR amplified three kinds of specificsequences of pathogens,combined with DHPLC amplified genotyping fragment.Meanwhile,PCR-DHPLC parameters were optimized for this process.48strains ofnon-pathogenic spirochetes were used to verify the sensitivity and specificity ofPCR–DHPLC for samples detection.For detection of antigen by enzyme-linkedimmunosorbent, we expressed out membrane protein A (OspA), which providedsubsequent preparation of colloidal gold strip. Finding relevant literature, OspAnumerous expressed in Borrelia burgdorferi infection bacteria, designed it primers atboth ends of restriction sites(NdeI/XhoI) to obtain sequence of the gene withrestriction sites.Double digestion the pET-28b vector with His tag,then ligated thedigested gene fragment.The recombinant expression vector pET-28b-rOspA wastransformed into the BL21(DE3),and the optimally expressional conditions offusion protein were determined by changing the concentration of the inducer, induction time and induction temperature.The concentration of IPTG inducedrecombinant protein on0.3mM,0.5mM and0.7mM,respectively.Induction time on3h、4h、5h, respectively.Induction temperature at20℃,25℃,30℃and37℃,respectively.SDS-PAGE method was applied to identify the expressed form ofpET-28b-rOspA protein and Western Blotting method was used to verify the fusionprotein specificity of pET-28b-rOspA.The obtained fusion protein was can bepurified by a nickel column to achieve optimal elution purposes by changing theconcentration of the eluate.Results:The length of amplification products were633bp,630bp,630bp by PCR withuniversal specific primers, and the sequencing results were completely correct bycomparing with Blast;The specific bands never found in the48kinds of non-pathogen specific Lyme strains. pathogen specificity of48kinds of non-applicationof Lyme strains of different species;the amplified fragments (without furtherprocessing) directly inject into DHPLC.The result indicates that the three kinds ofbacteria were entirely distinguished by DHPLC in partially denaturing conditions at55.8℃.Through the establishment of PCR-DHPLC genotyping method, the standardpatterns were established.The sensitivity of PCR-DHPLC method is0.01pg/μL onthe level of nucleic acid and distinguished the samples with high specificity.Therecombinant Borrelia burgdorferi gene sequences (pET-28b-rOspA) was successfullyconstructed and sequenced the result correct.The pET-28b-rOspA was transformedinto BL21(DE3) after double digestion.The best express conditions ofpET-28b-rOspA was determined to be0.5mM IPTG,37℃and4h induction bychanging the induction time,temperature and IPTG concentration.The recombinantprotein pET-28b-rOspA was found be an inclusion body by SDS-PAGE and verifiedby Western blotting.The recombinant protein was purified by Nickel column,achieved better result.Conclusions:This study successfully established the PCR-DHPLC standard map of three kind spirochete bacteria and constructed the recombinant pET-28b-rOspA fusionproteins,providing a foundation for Colloidal gold test strip.