Expression And Clinical Diagnostic Value Of Recombinant Treponema Pallidum Proteins Tp0100 And Tp1016

Background:Syphilis is a chronic systemic sexually transmitted disease cause by Treponema pallidum(Tp).The incidence of syphilis has been increasing year by year in China,but no effective vaccine can be used for syphilis prevention and treatment.Early laboratory diagnosis of syphilis is an important measure for the prevention and control of syphilis,mainly dependent on antibody detection.In recent years,serological methods based on recombinant T.pallidum proteins for the diagnosis of syphilis have become the mainstream of laboratory diagnosis of syphilis.However,these methods still have some defects in the diagnosis of early/late syphilis and the determination of therapeutic effect of syphilis.Hence,it is of great importance to seek novel recombinant antigens for syphilis serodiagnosis.Previous studies have shown that the Tp0100(thiorexin-like protein,TIP)and Tp1016(basic membrane protein,BMP)are reactive with the sera of patients with different stage syphilis and the sera of Tp-infected rabbits,suggesting their potential clinical value in the diagnosis of syphilis.Objective:The indirect enzyme-linked immunosorbent assay(ELISA)based on recombinant proteins Tp0100 and Tp1016 was established to evaluate the clinical value of these two recombinant proteins in serological diagnosis of syphilis,and to provide experimental basis for screening novel serological antigen for syphilis.Methods:(1)The recombinant vectors p ET28a-Tp0100 and p ET28a-Tp1016 were constructed and transformed into E.coli BL21(DE3).The expression of Tp0100 and Tp1016 were induced by IPTG and purified by Ni2~+-NTA column.Western blotting(WB)was used to identify the specificity and immunoreactivity with sera from Tp-infected rabbits.(3)ELISAs based on recombinant Tp0100(Tp0100-ELISA)or Tp1016(Tp1060-ELISA)were established to detect the specific antibodies in serum samples from patients with syphilis(340 cases),patients with non-syphilitic conditions(180 cases)and healthy persons(80 cases).Compared with T.pallidum particle agglutination assay(TPPA)and Toluidine red unheated serum test(TRUST),SPSS and Graph Pad PRISM statistical software were used to analyse the results from Tp0100-ELISA and Tp1060-ELISA to evaluate the potential value of Tp0100 and Tp1016 as candidate antigens for serologic diagnosis of syphilis.Results:(1)The size of gene fragments amplified by PCR was about615bp and 1038bp,which was consistent with the expected size;The recombinant plasmid vector was sequenced and confirmed to be the target gene sequence by BLAST comparison.(2)The molecular weights of the expressed proteins were 22k Da(Tp0100)and 39k Da(Tp1016),respectively.WB results showed that the two proteins could be specifically recognized by Tp-infected rabbit serum.(3)Using the results of TPPA method as the gold standard,the areas under the ROC curve of Tp0100-ELISA and Tp1016-ELISA were 0.99 and 0.869,respectively,indicating that the diagnostic effect of Tp0100-ELISA method was better,while the Tp1016-ELISA method was relatively poor.The cut-off values of Tp0100-ELISA and Tp1016-ELISA calculated by SPSS software indicate that,compared with those of the TPPA,the sensitivities of Tp0100-ELISA and Tp1016-ELISA were 97.35%and 75%respectively,and the specificities were 98.46%and 79.61%,respectively,resulting in consistencies of 97.83%and 77%,respectively.(6)The OD value of syphilis serum in different periods had a small overlap with the that of negative serum(normal and cross serum),indicating the existence of gray area.(7)The titer change of TRUST before and after treatment were compared with OD value change of Tp0100-ELISA and Tp1016-ELISA,respectively,showing that titer change of TRUST was positively correlated with OD value change of Tp0100-ELISA,and the correlation coefficient was 0.807(P<0.05).The correlation coefficient between TRUST and Tp1016-ELISA was 0.192(P>0.05),indicating no significant significance.Conclusions:Tp0100 is a promising antigen for serologic diagnosis and therapeutic evaluation of syphilis.Tp1016 is not suitable for serologic diagnosis of syphilis alone.