The Immunoprotection Of Recombinant Putative Treponema Pallidum Adherins Tp0225 And Tp0421 In Rabbit Model

Objective: To explore immunogenicity and immunoprotection of the recombinant putative Treponema pallidum(Tp)adhesins Tp0225(r Tp0225)and Tp0421(r Tp0421)in New Zealand rabbit model and to screen candidate vaccine molecules for syphilis.Methods:1.Expression and identification of recombinant proteins.E.coli.transformed with the recombinant prokaryotic vectors p ET-28a/ Tp0225 and p ET-28a/ Tp0421 were induced to express r Tp0225 and r Tp0421,verified by SDS-PAGE and Western blot,respectively.2.Animal immunity and immunogenicity detection of recombinant proteins.The male New Zealand rabbits were randomly divided into four groups: A: PBS group(negative control group),B: Freund’s adjuvant immunization group(blank control group),C: r Tp0225 immunization group,D: r Tp421 immunization group,and immunized with PBS,adjuvant,the purified adjuvant-added r Tp0225 or r Tp421 by intradermal and intramuscular injections for three times at two-week intervals.Two weeks after the last immunization,rabbit sera were collected to detect the titer of recombinant protein-specific Ig G antibody by ELISA,and rabbit spleen cells were collected to detect IFN-γ and IL-2 m RNA transcription level by reverse transcription PCR(RT-PCR).3.Tp challenge and evaluation of immune protection.After challenge with Treponema pallidum on the back of New Zealand rabbits,lesions were monitored daily for diameter and ulceration.After 21 th day post challenge,inflammatory cellular infiltration of cells in skin lesions were observed quantitative PCR(q PCR)was used to detect the Tp-DNA load of different organs and tissues.Results:1.SDS-PAGE and Western blot showed that recombinant E.coli expressed specific 25-k Da-(r Tp0225)and 70-k Da-size(r Tp0421)proteins,respectively.2.Two weeks after the last immunization,the titers of specific against-r Tp225 and against-r Tp421 Ig G antibodies were 1:25,600 and1:102,400,respectively.3.RT-q PCR results showed that the transcriptional levels of IFN-γand IL-2 in group C and D were significantly higher than those in group A(P<0.01)and group B(P<0.01),without significant difference between group A and group B.4.At day 15 post Tp challenge,there was no significant difference in the diameter of skin lesions among the four groups,but the ulcer formation rate of group C and D was lower than that of group A and B(P<0.01)at day 21 post Tp challenge,while there was no significant difference between control group A and B.5.The q PCR results revealed that there were higher Tp-DNA concentrations in skin lesions and blood,and lower in heart,liver,spleen,kidney,testis and lung.However,there was no significant difference in Tp-DNA concentrations in all tissues measured between the four groups.6.Histopathological results indicated inflammatory cellular infiltration in all the four groups,but the number of inflammatory cells in the C and D groups was significantly more than that in the group A and group B,without significant difference between group A and B.Conclusions:The recombinant Tp0225 and Tp0421 showed good immunogenicity.Immunization of r Tp0225 and r Tp0421 revealed a significant decrease in lesion ulceration in Tp-infected New Zealand rabbits,but could not prevent Tp infection and inhibit T.pallidum dissemination to the distant tissues in rabbits.