Expression And Preliminary Application In Clinical Syphilis Serodiagnosis Of Recombinant Protein Tp0463of Treponema Pallidum

Objective To express of recombinant protein Tp0463(rTp0463) of Treponema pallidum (Tp)by genetic engineering technology and to evaluate its preliminary application in clinical syphilisserodiagnosis for providing a basis for discovery of a novel candidate syphilis diagnostic antigen.Methods Gene sequence of Tp0463was obtained from GenBank and segment of Tp0463gene coding8thto110thamino acid residues was amplified by PCR using genomic DNA of T.pallidum Nichols strain as a template. Target gene was cloned into cloning T-A vectorpMD18-T and then subcloned into prokaryotic expression vector pET-28a(+) to construct arecombinant expression plasmid pET-28a(+)/Tp0463. E.coli BL21transformed withpET-28a(+)/Tp0463identified by PCR, double digestion and sequencing were induced to expressrecombinant proteins by IPTG induction. rTp0463were purified with Ni-NTA affinitychromatography. Purity and expression form of rTp0463protein were analysed by SDS-PAGE andBCA method was used to detect concentration of purified rTp0463. Western blot wasperformed to identify antigenicity and specificity of rTp0463. Indirect ELISA was established byusing purified rTp0463as a diagnostic antigen coated on microtiter plates. ELISA was then usedto test40standard positive and negative syphilis serum samples. Optimal ELISA method wasrandomly performed to detect150positive serum samples from syphilis patients and150negtiveserum samples from healthy persons or individuals with cross reaction with syphilis which wereconfirmed by TPPA method. Compare to gold standard TPPA, preliminary application of Tp0463in clinical syphilis serodiagnosis was evaluated.Results An approximate330bp-length fragment was amplified by PCR. The Tp0463genewas confirmed to be inserted into the prokaryotic recombinant pET-28a(+) by identification withPCR, double digestion and sequencing. The result of SDS-PAGE showed that E.coli BL21withpET-28a(+)/Tp0463were induced to express an approximate16KDa recombinant protein with adominant soluble form and more than95%purity. Western blot indicated that purified proteinswere able to be recognized specially by pooled sera from syphilis patients but not by pooled serafrom non-syphilis patients. The established indirect ELISA used for detection of standard positive and negative syphilis serum samples revealed a95.45%of positive coincidence rate,100%ofnegative coincidence rate and general coincidence rate of97.5%, respectively. Random test inclinical serum samples demonstrated, compared to TPPA, the ELISA indicated a sensibility of88.67%, pecificity of100%and general coincidence rate of95.0%, respectively.Conclusionsâ… . A recombinant expression plasmid pET-28a(+)/Tp0463was successfully constructed andthe recombinant protein Tp0463expressed efficiently in E.coli has good antigenicity.â…¡. Compared to gold standard TPPA method, the ELISA based on recombinant antigenTp0463showed a higher sensibility and very high pecificity, which indicates the recombinantprotein Tp0463has potential clinical diagnosis value but its clinical application should be furtherassessed.