Expression And Clinicalapplication Of Recombinant Treponema Pallidum Proteins Tp0727 And Tp0163

Background: Syphilis is a sexually transmitted disease caused by Treponema pallidum(Tp)infection,which seriously endangers human physical and mental health.However,no syphilis vaccine has been developed so far.Early diagnosis of syphilis has become an important measure for prevention and control of syphilis.Syphilis presents multistage development,with complex and diverse clinical manifestations,often needs the aid of syphilis laboratory diagnosis.In recent ten years,the serological method based on recombinant Tp antigen has greatly promoted the rapid development of the specific laboratory diagnosis of syphilis.However,the current recombinant antigens are still insufficient for the diagnosis of early/late syphilis,or the differentiation of different infection stages,or the judgment of efficacy.Previous studies have shown that Tp flagellum hook-based complex protein Tp0727 and ATP binding cassette transporter Tp0163 showed a strong immune response to the serafrom Tp-infected rabbits and syphilis patients,showing potential diagnostic value.Objective: To evaluate the diagnostic value of ELISA method based on recombinant proteins Tp0727 and Tp0163 on clinical samples,so as to provide experimental basis for the discovery of novel antigens for syphilis diagnosis.Methods:1.The whole gene sequences of Tp0727 and Tp0163 were queried from Genbank,and Tp0727 and Tp0163 genes were amplified by PCR with specific primers.The prokaryotic expression vectors pET28a-Tp0727 and pET28a-Tp0163 were constructed,and then were transformed into E.coli BL21(DE3)after sequencing.2.Tp0727 and Tp0163 recombinant proteins were expressed by IPTG induction,identified by Western Blot,and then purified by Ni2+-NTA affinity chromatography.Western Blot was used to detect the immunoreactivity and specificity of recombinant proteins Tp0727 and Tp0163 with sera from Tp-infected rabbits.3.Indirect ELISA methods based on recombinant proteins Tp0727 and Tp0163 were established to test the sera from syphilis patients(209cases),cross-reaction of immune sera(93 cases)and negative control sera(96 cases).The results were compared with those of TPPA and TRUST assays to evaluate the value of Tp0727-ELISA and Tp0163-ELISA inserological diagnosis of syphilis.Results:1.The sizes of the gene fragment amplified by PCR was about 1392 bp and 861 bp,which was consistent with the size of Tp0727 and Tp0163 gene,respectively.The inserted genes were confirmed to be the target gene by sequencing.2.The molecular weights of the target proteins expressed by the recombinant plasmids were 49kDa(Tp0727)and 35kDa(Tp0163),respectively.WB showed that Tp0727 and Tp0163 proteins were active specifically with Tp-infected rabbit sera.3.The ROC curve analysis results showed that the area under the ROC curve of TP0727-ELISA and TP0163-ELISA was 0.9930 and0.9751,respectively,with the TPPA detection results as the gold standard,indicating that the two methods had better diagnostic effects.4.Tp0727-ELISA and Tp0163-ELISA were used to detect the specific Tp-IgG in clinical samples,and the coincidence rates with TPPA were relatively high,which were 95.0% and 91.7%,respectively.The sensitivity was 91.9% and 88.0%,and the specificity was 98.4% and95.8%,respectively.The correlation coefficients between the change of OD value and the change of TRUST titer by Tp0727-ELISA and Tp0163-ELISA were 0.705 and 0.735,respectively(P<0.05),indicating that there was a certain correlation between the two ELISA methods andTRUST method.Conclusions: Tp0727 and Tp0163 have potential value in serological diagnosis of syphilis and are expected to be candidate antigens for diagnosis of syphilis.