| Objective: To evaluate the immunoprotective potential and diagnostic value of recombinant Treponema pallidum(Tp)adhesin Tp0750(rTp0750)in New Zealand rabbits and to screen novel candidate syphilis vaccine molecules and diagnostic antigens.Methods:1.The prokaryotic expression vector pET-28 a /Tp0750 was constructed and induced to express rTp0750 in Escherichia coli and rTp0750 was identified by Western blot.2.New Zealand rabbits were randomly divided into three groups: A: rTp0750(experimental group),B: rTp0751(positive control group)and C: PBS(negative control group).Purified recombinant proteins or PBS were injected subcutaneously and intramuscularly into New Zealand rabbits for four times at two weeks intervals.Sera were collected to detect specific IgG antibody levels before each immunization.Spleen cells were collected 2 weeks after the last immunization,and the proliferation of spleen lymphocytes and the mRNA transcription level of IFN-? were detected by CCK-8 cell proliferation kit and qPCR kit,respectively.Meanwhile,rabbit's backs were and injected intradermally with alive T.pallidum(Nichols strain)and changes in skin lesion diameter and formation of skin ulcer were observed,and Tp-DNA burden of different tissues of New Zealand rabbits was detected by real-time fluorescent quantitative PCR(qPCR).Inflammatory cell infiltration in pathological tissue of skin lesions and renal were observed under light microscope.3.An indirect rTp0750-based ELISA was established to detect specific IgG antibody levels in sera from syphilis patients at different stages,normal sera and cross sera and to evaluate the diagnostic value of rTp0750.Results:1.Western blot results showed that the constructed prokaryotic expression plasmid pET-28a/Tp0750 expressed a specific protein with a molecular weight of 25 kDa.2.In group A and B,the corresponding specific antibody appeared in the second week after immunization and gradually increased,reaching a peak in the eighth week(P<0.01).At this time,the specific IgG titer of group B was higher than that of group A(P<0.05).3.CCK-8 results showed that the stimulating index(SI)of rabbit lymphocytes in group A and group B was significantly higher than that in group C(P<0.01),but there was no significant difference between group A and group B.RT-PCR results showed that the transcription level of IFN-? of rabbit spleen cells in group A and group B was significantly higher than that in group C(P<0.01),and that in group B was higher than that in group A(P<0.05).4.On the 14 th day post challenge,the skin loss diameter of group A and group B was smaller than that of group C,while that of group B was smaller than that of group A.On the 21 th day post challenge,the incidence of skin ulcer was lower in group A and group B than in group C,and lower in group B than in group A.5.The results of qPCR showed that the most Tp load was in the skin and blood.There was no significant difference between the three groups in the Tp load in the skin lesions,but the Tp load in the liver,spleen,blood and kidney tissues of the rabbits in group A and group B was significantly less than that in group C(P<0.01),and there was no significant difference between group A and group B.6.Histopathological results showed that inflammatory cell infiltration was observed in the skin lesions of the three groups,but the number of inflammatory cells in group C was less than that in group A and group B,and there was no significant difference between group A and group B.There was obvious inflammatory cell infiltration in the kidney of group C,but not in group A and group B.7.The sensitivity and specificity of Tp0750-ELISA were 51.7% and 95.2%,respectively.Conclusion:1.Tp0750 induced high levels of specific antibodies and cellular immune responses in New Zealand rabbits.2.Tp0750 immunization attenuates lesion development and inhibits T.pallidum dissemination in New Zealand rabbits.3.Tp0750 has high diagnostic specificity but low sensitivity,so it is not suitable for the serological diagnosis antigen of syphilis. |
PDF Full Text Request