Identification of in vivo-induced antigen Tp0462 of Treponema pallidum and evaluation of its application to serodiagnosis of syphilis

Abstract

Objective To screen and identify the in vivo-induced antigen Tp0462 of Treponema pallidum (Tp) , and to evaluate its value for clinical serological diagnosis of syphilis. Methods Genome-wide DNA was extracted from the Tp Nichols strain, and polymerase chain reaction (PCR) was performed to amplify the Tp0462 gene. A recombinant plasmid pET30a (+) -Tp0462 was constructed and transfected into the Escherichia coli (E.coli) Rosetta (DE3) strain. Recombinant protein Tp0462 was abundantly expressed, purified and identified. A total of 18 New Zealand rabbits were randomly and equally divided into 3 groups: viable Tp-incubating group incubated with viable Tp in the testes, inactived Tp-incubating group incubated with ultraviolet irradiation-killed Tp in the testes, and control group receiving no treatment. After incubation, blood samples were collected at different time points, and the sera were isolated for identification of characteristics of in vivo-induced antigen Tp0462. Enzyme-linked immunosorbent assay for Tp0462 (Tp0462-ELISA) , Treponema pallidum particle agglutination assay (TPPA) , preliminary syphilis screening ELISA and rapid plasma reagin (RPR) card test were applied in 336 clinical serum samples from patients with syphilis, so as to preliminarily evaluate the value of Tp0462 for the diagnosis of syphilis. Results The optimum conditions for expression of the recombinant plasmid Tp0462-pET30a (+) in E.coli Rosetta (DE3) strains were the treatment with 0.5 mmol/L isopropyl thiogalactoside (IPTG) on a shaker at 180 rpm for 4 hours. In the viable Tp-incubating group, the serum level of specific anti-Tp0462 antibody sharply increased from week 2, and went steady after week 5. However, the specific anti-Tp0462 antibody maintained a low level in the inactived Tp-incubating group and the control group. The viable Tp-incubating group showed a significantly higher level of specific anti-Tp0462 antibody compared with the inactived Tp-incubating group and the control group (both P < 0.05) , while no significant difference was observed between the inactived Tp-incubating group and the control group (P = 0.256) . The level of anti-Tp92 antibody was significantly higher in the viable Tp-incubating group and the inactived Tp-incubating group than in the control group (P < 0.05) , while there was no significant difference between the viable Tp-incubating group and the inactived Tp-incubating group (P = 0.127) . Compared with TPPA, the sensitivity, specificity, consistency rate and area under the curve (AUC) of Tp0462-ELISA for the diagnosis of syphilis were 91.7%, 98.8%, 95.2% and 0.997 respectively. Tp0462-ELISA was consistent to preliminary syphilis screening ELISA and RPR with a Kappa coefficient of 0.846 and 0.293, respectively. Conclusion Tp0462-ELISA has shown evidently higher sensitivity and specificity in the serodiagnosis of syphilis, and Tp0462 can serve as promising antigens for the diagnosis of syphilis. Key words: Syphilis; Treponema pallidum; Syphilis serodiagnosis; In vivo induced antigen; Tp0462