Preparation And Immunocompetence Of Recombinant E.coli Ghost Expressing Treponema Pallidum Adhesin Tp0751

[Objective] To construct the recombinant E. coli bacterial ghost(EBG) of Treponema pallidum adhesin Tp0751(r Tp0751-EBG) and to lay the foundation to further investgate its potential immunoprotection against T. pallidum(Tp) infection. [Methods](1) Preparation and identification of EBG. E. coli DH5α transformed with the plasmids p HH43 containing phage phi X174 lysis gene E was induced by temperature control to produce BG. The lysis rate was calculated and BGs were identified by agarose gel electrophoresis of DNA and Transmission Electron Microscope(TEM).(2)Construction and expression of the vector containing the membrane anchor gene E’. The PCR-amplified membrane anchor gene E’ was cloned into p ET28a-Tp0751 with target gene Tp0751, to form expression vector p ET28a-E’-Tp0751. The vectors were transformed into E. coli BL21 to be induced to express E’-Tp0751 confirmed by WB.(3)Construction of recombinant expression plasmid of r Tp0751-EBG. The enzyme-digested p HH43 with E-box and p ET-32 a were linked to form p ET32a-E-box. The enzyme-digested p ET32a-E-box and p ET28a-E’-Tp0751 were then construct into p ET-28a-E’-Tp0751-E-box.(4)Expression and identification of r Tp0751-EBG. p ET-28a-E’-Tp0751-E-box was transformed into E. coli BL21 to express Tp0751 at 28 ℃and produce BGs at 42℃.The lysis rate was calculated and BGs were identified by electrophoresis and TEM.(5)Animal immunization. r Tp0751-EBG, Tp0751, BG and PBS were immunized into BALb/c mice intramuscularly or intranasally for 3 times. Before each immunization and 10 days after last boost, sera and genital tract lavage fluid were collected, mice spleen cells were prepared.(6)Determination of immunocompetence of recombinant Tp0751-BG. Indirect ELISA was used to detect levels of Ig G or s Ig A against Tp0751 in sera or genital tract lavage fluid. CCK-8 assay was performed to test proliferation of splenic lymphocyte. Indirect ELISA was used to detect levels of IFN-γ produced by splenic lymphocyte. [Results](1)Preparation and identification of EBG At 42℃, E gene in p HH43 was activated to result in lysis of E. coli and formation of BGs. The lysis rate is 98.13% No DNA bands were observed in agarose electrophoresis. Lysis tunnel formation and expulsion of the cytoplasmic contents were viewed by TEM but BGs still remained basic morphological shapes.(2)Construction and expression of the vector with membrane anchor gene E’ A band with about 180-bp-size was amplified by PCR. A protein with about 28-KDa-size was expressed in soluble form. WB showed that the recombinant proteins were active with Tp-infected rabbit sera.(3)Construction, expression and identification of r Tp0751-EBG plasmid. The results of enzyme digestion and sequencing showed that p ET-28a-E’-Tp0751-E-box was constructed successfully. At 28℃, a protein with about 28-KDa size was expressed and conformed to be active with Tp-infected rabbit sera. At 42℃, D600 value began to decline. The lysis rate is 96.37%. The results of agarose electrophoresis and TEM were similar with those of EBG.(4)Levels of systemic and mucosal humoral immune response induced by r Tp0751-EBG. Levels of specific Ig G in sera from r Tp0751-EBG(i.m. and i.n.) and Tp0751 groups increased gradually, reaching the peak at the 4th week post immulization, with titers of ≥1:25600 titers, and were significantly higher than that from EBG and PBS groups. There no obvious differences between the former three groups. Levels of specific s Ig A in genital tract from Tp0751-BG(i.m. and i.n.) groups increased gradually, reaching the peak at the 4th week post immulization, with titers of 1:6400, and were significantly higher than that from r Tp0751, EBG and PBS groups. There no obvious differences between the former two groups.(5) Levels of cellular immune response induced by r Tp0751-EBG. Stimulate Index(SI) in Tp0751-BG(i.m. and i.n.) and Tp0751 groups were significantly higher than that in EBG and PBS groups. There no obvious differences between the former three groups. Levels of IFN-γ groups were significantly higher than that in r Tp0751(P<0.05) EBG and PBS groups(P<0.01). There no obvious differences between the former two groups. [Conclusions](1) Recombinant EBGs of Treponema pallidum adhesin Tp0751 were constructed successfully.(2) Recombinant Tp0751-BG can induce mice C57BL/6 to produce stronger mucosal and systemic humoral immune response and systemic cellular immune response.