Expression And Preliminary Clinical Application Of Candidate Diagnostic Antigens Of Treponema Pallidum

Objective: To assess the significance of recombinant proteins Tp0608, Tp0965andTp1038(TpF1) of Treponema pallidum (Tp) in the serodiagnosis of syphilis, and toprovide an experimental basis for discovery of novel candidate syphilis diagnosticantigens.Methods: Gene sequences of Tp0608and Tp0965were obtained from GenBank andfull length of the two genes were amplified by PCR using genomic DNA of T. pallidumNichols strain as a template. Then target fragments digested by enzyme were clonedinto the prokaryotic expression vector pET28a(+) to construct the recombinant vectorspET28a(+)-Tp0608and pET28a(+)-Tp0965.After identified by double digestion andsequencing, recombinant vectors then were transformed into E.coli BL21(DE3) forprotein expression by IPTG induction.Expression form and antigenicity of recombinantproteins were analysed by SDS-PAGE and Western blot, respectively. Recombinantproteins were purified with Ni-NTA affinity chromatography and SDS-PAGE wasperformed to analyse purity.Indirect ELISA was established by using single orcombined purified rTp0608, rTp0965and rTpF1as diagnostic antigens coated onmicrotiter plates, with positive syphilitic serum or normal serum as the primaryantibodies, and HRP-conjugated goat anti-human IgG as secondary antibodies.Concentration of coated antigens, combination of antigens and its concentration,dilution of sera(primary antibody) were optimized in order to screen the optimalELISA. The optimized rTpF1-rTp0965-ELISA was performed to determinateconfirmed syphilitic sera and normal sera and sensitivity, specificity and consistencywere analysed statistically. This optimal ELISA method was further used to randomlydetect TPPA-positive serum samples from syphilis patients and TPPA-negtive serumsamples from healthy persons or individuals with cross reaction with syphilis.Compare to TPPA and domestic Tp-ELISA kit, preliminary application of rTpF1-rTp0965-ELISA in clinical syphilis serodiagnosis was evaluated.Results:Ⅰ. Approximate888bp-length and960bp-length fragments were amplified by PCR.The Tp0608and Tp0965genes were confirmed to be inserted into the prokaryoticrecombinant pET28a(+) by identification with double digestion and sequencing whichwere proved to be consistent with published sequences in GenBank.Ⅱ. The recombinant plasmids pET28a-Tp0608and pET28a-Tp0965transformed intoE.coli BL21(DE3) were induced by IPTG to express about38kDa and43kDarecombinant proteins, with inclusion body and soluble form, respetively.Ⅲ. After purified by Ni2+-NTA affinity chromatography pillar, the purity of bothrTp0608and rTp0965was above95%; Western blot results showed that the rTp0608and rTp0965were only able to be recognized specially by sera from syphilis patientsbut not by sera from non-syphilis patients.Ⅳ. ELISA showed that theoptimal single antigen was TpF1(4μg/mL）and the optimalcombined antigens were TpF1(4μg/mL) and Tp0965(4μg/mL), with highest A value(P/N). The optimal serum dilution was1:200. Repeatability tests indicated that theaverage coefficient of variation(CV) of optimized rTpF1-rTp0965-ELISA was2.6%(less than10%).Ⅴ. The established indirect ELISA used for detection of confirmed31positive and19negative syphilis serum samples revealed a96.7%of susceptibility,100%ofspecificity and general coincidence rate of98.0%, respectively. Random test in93clinical serum samples demonstrated, compared to TPPA, the ELISA indicated asensibility of97.6%, pecificity of100%and general coincidence rate of97.6%,respectively. Compared to Tp-ELISA, rTpF1-rTp0965-ELISA showed similarsusceptibility but higher specificity.Conclusion:Ⅰ. The recombinant proteins Tp0608and Tp0965were successfully expressed, bothof which have good antigenicity.Ⅱ. Recombinant proteins TpF1and Tp0965, but not Tp0608are expected to becomecandidate diagnostic antigens. The established rTpF1-rTp0965-ELISA reveals highspecificity, higher sensitivity and repeatability but its clinical application needs furtherevaluation and repeat test.