Construction And Immunogenicity Of Recombinant E.coli Ghosts Expressing Trivalent Treponema Pallidum Adhesions

Objective:The aim of this study is to construct the recombinant prokaryotic and eukaryotic E.coli bacteria ghost(EBG)expressing trivalent adhesions Tp0751-Tp0136~N-Tp0435 of Treponema pallidum to efficiently induce humoral and cellular immune responses in BALB/c mice,and provide a potential novel method for syphilis vaccine developing.Methods:The recombinant prokaryotic expression plasmids of pET30a-E'-Tp0751-Tpr0136~N-Tp0435 were constructed and transformed it into E.coli BL21 to express fusion protein Tp0751-Tpr0136~N-Tp0435.Recombinant prokaryotic E.coli bacterial ghosts(EBGs)were generated by chemically-induced lysis(chemical osmotic pressure).Agarose gel electrophoresis was used to identify the recombinant EBGs and Western Blot(WB)was used to identify the expression of the target protein.The recombinanteukaryoticexpressionplasmidsofpcDNA3.1(+)-Tp0751-Tp0136~N-Tp0435(pcD-Q)were also constructed and transformed into the empty EBGs to create recombinant DNA ghosts.After48h'treatmentwithrecombinantDNAEBGs,rTp0751-Tpr0136N-Tp0435 expressed in RAW264.7 cells were tested by WB.For immunocompetence in mice,the female BALB/c mice were randomly divided into 7 groups,including three control groups,A(PBS),B(EBG),C(empty pcD),and four experimental groups,D(naked pcD/Q),E(pCD-Q/BG),F(E'-Q-BG)and G(pCD-Q/BG+E'-Q-BG)(Q/Q=Tp0751-Tp0136~N-Tp0435 protein/protein-coding gene).All the mice were immunized for three times by intramuscular injection at two weeks intervals.At weeks 2,4,6 post primary immunization,the levels of specific IgG/IgG1/IgG2a in sera and SIgA in genital tract lavage fluid were measured by ELISA.At week 2 post last boost,levels of lymphocyte proliferation and IFN-?secretion in spleen cells were measured by CCK-8 Cell Counting Kit and ELISA as well,respectively.Results:Therecombinantprokaryotictargetprotein Tp0751-Tp0136~N-Tp0435 expressed in EBGs was active with Tp-infected rabbit sera by WB.The lysis rate of EBGs is more than 98%.The agarose electrophoresis gel showed that there was no nucleic acid in the cytoplasm of EBGs.The loading rate of pcD-Q to empty EBGs was74.7%.The recombinant eukaryotic target protein expressed in transfected RAW264.7 cells was active with Tp-infected rabbit sera.The titers of specific IgG,IgG1,IgG2a and SIgA in experimental group D,E,F,G gradually increased to significantly higher level as compared to the control groups(P<0.01),which reached its peak at week 6 after primary immunization(the titers of IgG,IgG1,IgG2a and SIgA were 1:51,200?1:51,200?1:6400 and 1:1600 in group G,respectively).Higher levels of specific IgG,IgG1,IgG2a were observed in group E,F,G as compared to group D(P<0.01),with group G higher than groups E and F after last boost(P<0.05).No obvious differences were observed between groups E and F.Higher levels of specific SIgA were observed in group E,F,G as compared to group D(P<0.01),No obvious differences were observed between group E,F and G.The rate of IgG2a/IgG1 was less than 1.0 in all experimental groups.At week 2 after the last boost,the stimulation index(SI)and levels of IFN-?in all experimental groups were all significant higher than the control groups(P<0.01),with group E,F and G higher than group D(P<0.01),and group G higher than group E and F(P<0.05).No obvious differences were observed between group E and F.Conclusions1.The recombinant prokaryotic and eukaryotic E.coli bacterial ghosts expressing trivalent adhesins Tp0751-Tp0136~N-Tp0435 are constructed successfully.2.The trivalent prokaryotic and eukaryotic EBGs can efficiently induce a Th2-biased mixed Th1/Th2 type systemic immune response and local mucosal response in mice.The heterologous boost could be more efficient than homologous boost during immunization process.