Gene cloning and expression of the Tp0453 antigen immuno-dominant epitope fragment of Treponema pallidum and its potential use in serodiagnosis of syphilis / 中华传染病杂志

ABSTRACT

Objective To clone and express the Tp0453 antigen immuno-dominant epitope fragment of Treponema pallidum (Tp) in Escherichia coli,in an effort to develop serological tests with increased specificity for the diagnosis of syphilis.Methods The gene encoding Tp0453 recombinant outer membrane protein fragment was amplified by polymerase chain reaction (PCR),and inserted into expression vector pQE30 after T-A cloning,then confirmed by restriction map.The constructed recombinant plasmid pQE30-Tp0453 was transformed to E.coli M15 for expression induced by isopropyl β-D-1-thiogalactopyranoside.The expressed product was identified by Western blot,and purified by Ni2+-NTA agarose column chromatography.A double antigen sandwich enzymelinked immunosorbent assays (ELISA) was established by using the recombinant Tp0453 protein to test sera from 48 patients with positive Treponema pallidum particle agglutination test (TPPA),and 40 negative sera as control.Results The PCR amplicon of the target gene was about 490 bp.The recombinant plasmid pQE30-Tp0453 was correctly constructed and successfully expressed in E.coli M15.The expressed product,with a relative molecular of about 21 000,existed in a form of inclusion body,accounting for about 18％ of total somatic protein,and reached a purity of more than 95％ after purification.Western blot showed specific reaction of the expressed protein with Tp positive serum.The ELISA tests with the 88 clinical samples yielded a sensitivity of 97.9％ (47/48),and specificity of 100.0 ％ (40/40).The consistency of results between the ELISA test and the TPPA test was 98.9 ％ (87/88).Conclusion The expressed Tp0453 fragment has showed good immunoreactivity with serum from patients with syphilis,providing the foundation of further development of serological diagnostic kit with increased specificity for the diagnosis of TP infection.