Screening,Preparation Of Treponema Pallidum Specific Diagnostic Antigen And Evaluation Of Clinical Application

ObjectiveSyphilis is a very harmful sexually transmitted disease of human over the world that is caused by infection with the Treponema pallidum subsp.pallidum(T.pallidum). Enzyme-linked immunosorbent assay(ELISA) based on genetic engineering antigen has the advantage of high-speed, easily handing and low cost, and has been used in clinica extensiviely. However, the current testing assay of syphilis does not present the satisfied sensitivity and specificity. In this research, we identified multitude Treponema pallidum specific outer membrane antigens based on bioimformatic analysis, and expressed recombinant protein by genetic engineering technique to provide the basis of theory and practice for the development of quick diagnostic ELISA kit applying to detect T.pallidum infection.Methods1. Treponema pallidum specific outer membrane antigens that may having good diagnostic potentiality for T.pallidum infection were selected according to other studies, and the immuno-dominant epitope of these protein was identified using bioimformatic software DNAstar.2. The gene coding TP17, TP0453 and TP0684 were amplified from T.pallidum complete genome by polymerase chain reaction(PCR) respectively, and the PCR product was cloned into prokaryotic expression vector to generate recombinant plasmid pET22b(+)-TP17, pET-28a(+)-TP0453, pET22b(+)-TP0684. The recombinant vectors were correspondly transformed and expreesed in E.coli BL21(DE3). The recombinant protein were purified with AKTA-explore 100 system. The expression products were analyzed by SDS-PAGE.Western-blot was used to test the immunocompetence of TP17,TP0453 and TP0684 antigens.3. Development of indirect ELISA assay. The puried TP17, TP0453, TP0684 protein were coated alonely or combinely. The optimal antigen coating combination and dose, the mode of antigen adsorbing, blocking buffer and blocking time, serum reacting time and temperature, goat anti-human IgG working concentration, coloration buffer were defined.4. Detection of clinical TP serum sample and country quality control serum by using the above established indirect ELISA.Results1. TP17, TP0453, TP0684 were selected as diagnostic antigen for detecting T.pallidum infection, and TPN17(20~156aa), TP0453(29~287aa), TP0684(30~434aa) were estimated as the immuno-dominant epitope of these protein respectively.2. Constructed the high performanceing expression vector pET22b(+)-TP17, pET-28a(+)-TP0453 and pET22b(+)-TP0684. The results of digestion with restriction DNA enzymes and sequencing of the recombinant plasmids showed the gene was constructed successfully .3. The recombinant plasmid were transformed in E.coli BL21(DE3) and induced with IPTG at 37℃. SDS-PAGE analysis showed that the relative molecule mass(Mr) of expressed target protein TP17, TP0453, TP0684 were 18kDa, 29kDa, 43kDa respectivily ,and the expressing ratio was indicated as 18%, 24% and 32% of total bacterial protein by UVP scan. The expression form of TP17 was proved to be supernatant: TP0453, TP0684 to be inclusion body.4. After purification by AKTA-explore , we got the target protein TP17 with purity of more than 95%; TP0453 with purity of more than 90% and TP0684 with purity of more than 85%.5. Western blot proved that the recombinant protein TP17, TP0453, TP0684 can specifically react with T.pallidum IgG positive sera,but no with negatine sera.6. Established and optimized indirect ELISA using different purified antigen. The optimal antigen combinant was TP17 plus TP0453, with coating concentration of 0.5μg/ml and 1μg/ml respectively; the optimal condition is 37℃4h, and then 4℃to stay overnight; the optimal blocking condition was determined as 1% BSA at 37℃for 2 hours followed by 4℃overnight; the serum reaction condition is 37℃45 min; the dilution of second antibody was 1:15000; the optimal coloration buffer was TMB.7. By using the above established ELISA, we examined the clinical TP serum sample and country quality control serum. The results showed 94.4% of sensitivity; 97.1% of specificity. The reproducibility was very high. The kit established by present study had good consitency with TPPA and existing domestic-made ELISA kit.Conclusion1. estimated the immuno-dominant epitope of TP17, TP0453, TP0684 protein using bioimformatic software DNAstar.2. Constructed the high performanceing expression vector pET22b(+)-TP17, pET- 28a(+)-TP0453 and pET22b(+)-TP0684.3. After purification by AKTA-explore ,we got the target protein TP17 with purity of more than 95%; TP0453 with purity of more than 90% and TP0684 with purity of more than 85%.4. Established and optimized indirect ELISA using purified TP17, TP0453, TP0684 antigen. The examination results showed that our method had high specificity and sensitivity. Our study provide the basis of theory and practice for the development of quick diagnostic ELISA kit applying to detect T.pallidum infection.5. Western blot proved Tp0684 has good immunoreactivity which can specifically react with T.pallidum IgG positive sera. But the diagnostic value of this protein need to be explored in ELISA intensively.