Preliminary Study On Immune Protective Effect Of Treponema Pallidum Lipoprotein Tpn32

Objective:In order to verify the vaccine potential of recombinant protein Tpn32 and screen new candidate of Treponema pallidum vaccine molecules,we evaluated the cellular immune response and humoral immune response induced by the immunization of Treponema pallidum membrane lipoprotein Tpn32 in New Zealand rabbits,as well as the immune protective effect of retard the progression of disease after Treponema pallidum attack.Methods: 1.The prokaryotic expression vector p ET-28a/ Tpn32 was constructed and verified.The constructed recombinant plasmid p ET-28a/Tpn32 was transformed into Escherichia coli for induction and expression,and the purified recombinant protein of Tpn32 was identified by Western Blot.2.New Zealand rabbits can be divided into three groups at random,r Tpn32 protein group(5),the PBS group(3)and adjuvant group(3).Then,r Tpn32,PBS or Freund’s adjuvant were injected intramuscularly and subcutaneously into New Zealand rabbits and the rabbits were immunized once every two weeks for 3 times in total.Venous blood from the ear margin of New Zealand rabbits was collected before each immunization,and the specific antibody level was detected after serum separation.The serum levels of IFN-γ,IL-6 and IL-8 were detected by IFN-γ ELISA kit,IL-6 and IL-8 ELISA kit,respectively,from New Zealand rabbits 2 weeks after last immunization.3.Two weeks after the last immunization,New Zealand rabbits were infected with Tp,and the appearance time and diameter of the lesions on the back of New Zealand rabbits were observed and recorded.Then Treponema pallidum load in New Zealand rabbit different tissues were detected through the real-time fluorescent quantitative PCR,and inflammatory cells infiltration in testicular tissue and skin lesions were verified by pathological dyeing.Results: 1.The successful construction of the prokaryotic expression vector of p ET-28a/Tpn32 was verified by PCR identification and sequencing results of recombinant plasmid p ET-28a/TPN32;2.Western Blot showed that the recombinant strain p ET-28a/TPN32 successfully expressed a specific protein with a molecular weight of 32 k Da.3.Compared with the PBS group and adjuvant,Tpn32 immune protein group began to produce specific antibodies in 2 weeks after immunization,and with the time increasing,the number of immune antibody levels also increased,at the 6th week,the Ig G antibody level of the protein-immunized group reached 1:125600,and the specific antibody level of the r Tpn32 immunized group was significantly different from that of the PBS group and the adjuvant group;4.The results of rabbit IFN-γ ELISA kit showed that the secretion level of IFN-γ in Tpn32 protein immunized group was significantly higher than that in PBS group(P<0.0001)and adjuvant group,there was no significant difference in IFN-γ secretion between PBS group and adjuvant group;5.According to the results of rabbit IL-6 ELISA kit and rabbit IL-8 ELISA kit,the levels of IL-8 in r Tpn32 protein immunization group were significantly higher than those in PBS group and adjuvant group;6.On day 9,lesions were present on all PBS and adjuvant animals,whereas the animals immunized with r Tpn32 protein group matched the level of lesions observed until days 12.After 21 days of T.pallidum infection,r Tpn32 protein infection site in New Zealand rabbits skin diameter size immune group was obviously less than the PBS group and adjuvant,and PBS and adjuvant group of New Zealand rabbit skin ulcer rate is obviously greater than r Tpn32 immune group;7.Real-time quantitative PCR results showed that the T.pallidum load in the infected site of New Zealand rabbits in the r Tpn32 immunization group generated no difference from that in the PBS group and the adjuvant group,while the T.pallidum load in the distal tissues,such as blood and testis of New Zealand rabbits in the r Tpn32 immunization group was significantly lower than that in the PBS group and the adjuvant group(P<0.01);8.A large number of inflammatory cell infiltrates were observed in the pathological sections of the dorsum lesions of New Zealand rabbits in the r Tpn32 immunization group;however,compared with the PBS group and the adjuvant group,the r Tpn32 immunized group had less inflammatory cell infiltration,such as lymphocytes and macrophages.Conclusions: 1.Immunization of r Tpn32 protein induced a higher level of specific Ig G antibody and Th1 cytokine IFN-γ in New Zealand rabbits;2.Immunization of New Zealand rabbits with r Tpn32 may slow down the development of dorsal lesions and delay the spread of Treponema pallidum to distal organs in vivo.