梅毒螺旋体黏附素Tp0751核酸菌影的构建及免疫原性研究

Construction and immunocompetence of recombinant DNA E. coli ghosts expressing Treponema pallidum adhesin Tp0751

目的:构建梅毒螺旋体(Tp)黏附素Tp0751的重组真核大肠埃希菌菌影(EBG)并检测其在免疫鼠中的免疫原性,为探讨新型梅毒疫苗奠定基础。方法:构建pc DNA3.1(+)/Tp0751真核表达载体,将其装载入已构建的空EBG中,形成重组核酸菌影pc D/Tp0751-BG,计算装载率;将核酸菌影转染鼠源性巨噬细胞RAW264.7,Western blot(WB)鉴定目的蛋白表达。将雌性BALB/c鼠随机分为A(PBS)、B(空EBG)、C(空pc DNA3.1)三个对照组和D(pc D/Tp0751)、E(pc D/Tp0751-BG)、F(pc D/Tp0751-BG+r Tp0751)三个实验组,各组间隔两周肌注免疫共三次,检测特异性血清Ig G及生殖道黏膜SIg A、小鼠脾细胞增殖水平和分泌IFN-γ水平。结果:重组真核质粒对菌影的装载率为76.1%;WB显示此转染细胞能有效表达重组目的蛋白。D、E、F实验组小鼠特异性血清Ig G与生殖道SIg A效价均随免疫次数增加而增加,各时间点均显著高于三个对照组(P<0.01),于末次加免后第8周达到峰值,此时F组Ig G与SIg A效价分别为1∶102 400与1∶12 800;首次加免2周后,E、F组均显著高于D组(P<0.01);末次加免2周后,F组显著高于E组(P<0.01)。末次加免后第8周,D、E、F组的刺激指数(SI)值与IFN-γ水平均分别显著高于三个对照组(P<0.01);E、F组均分别显著高于D组(P<0.01);F组分别均高于E组(P<0.05)。结论:Tp0751真核质粒菌影具有良好的免疫原性,在小鼠体内诱生了有效的系统和黏膜的体液应答以及系统细胞免疫应答;异源加免较同源加免免疫效果更好。

Objective:To construct the recombinant DNA E.coli ghosts(EBGs)expressing Treponema pallidum adhesin Tp0751(pcD/Tp0751-BG)and determine its immunocompetence in immunized mice,and provide a potential novel method for syphilis vaccine developing.Methods:The recombinant eukaryotic expression plasmid pcDNA3.1(+)/Tp0751 was constructed and loaded into empty EBGs to create pcD/Tp0751-BG.The loading rate was determined accordingly.Macrophages cell line RAW264.7 was transfected with pcD/Tp0751-BG,and the expression of recombinant Tp0751(rTp0751)protein was detected by Western blot(WB).For immunocompetence in mice,the female BALB/c mice were randomly divided into 6 groups,including three control groups,A(PBS),B(EBG),C(empty pcDNA),and experimental group D(naked pcD/Tp0751),E(pcD/Tp0751-BG)and F(pcD/Tp0751-BG+rTp0751).All the mice were immunized as indicated for three times by intramuscular injection at two weeks intervals.The levels of specific IgG in sera and SIgA in genital tract lavage fluid were measured by ELISA.Levels of lymphocyte proliferation and IFN-γsecretion in spleen cells were measured by CCK-8 Cell Counting Kit and ELISA as well,respectively.Results:The loading rate of pcD/Tp0751 to EBGs was 76.1%.WB showed that the target recombinant protein pcD/Tp0751 expressed in RAW264.7 cells was active with Tp-infected rabbit sera.The titers of specific IgG and SIgA in group D,E,F gradually increased to significantly higher level as compared to the control groups(P<0.01),which reached its peak at wk 8 after last immunization(the titers of IgG and SIgA were 1∶102 400 and 1∶12 800 in group F,respectively).Higher levels of specific IgG and SIgA were observed in groups E and F as compared to group D after first boost(P<0.01),with groups F higher than group E after last boost(P<0.01).At wk 8 after the last boost,the stimulation index(SI)and levels of IFN-γin group D,E,F were all significant higher than the control groups(P<0.01),with group E and F higher than group D(P<0.01),and group E higher than group F(P<0.05).Conclusion:The recombinant DNA EBGs of T.pallidum adhesin Tp0751(pcD/Tp0751-BG)possesses the immunocompetence to induce not only strong mucosal and systemic humoral immune response but also systemic cellular immune response in BALB/c mice.The heterologous boost can be more efficient than homologous boost during immunization process.