梅毒螺旋体融合双价DNA疫苗的构建及其免疫活性研究

目的 构建含有梅毒螺旋体Gpd和Tp92抗原编码基因的真核表达重组体,并检测其在兔体内的免疫应答效果。方法 定向克隆构建双基因融合真核表达重组体pcDNA3.1(+)/Gpd-Tp92,用免疫组化技术检测其在HeLa细胞中的表达;同时将其与先期构建的pcDNA3.1(+)/Gpd、pcDNA3.1(+)/Tp92真核表达重组体分别免疫新西兰兔,ELISA检测免疫兔特异性抗体产生水平和脾细胞IFN-γ诱导水平,MTT法检测兔脾细胞增殖水平。结果 pcDNA3.1(+)/Gpd-Tp92在HeLa细胞中能有效表达。新西兰兔分别接种3种核酸疫苗后,均能产生特异性抗体,第3次免疫后2周抗体最高滴度均可达1:1024及以上,免疫后兔脾细胞受相应蛋白刺激有明显增殖反应,细胞培养上清中IFN-γ水平显著升高。检测指标均显著高于空质粒对照组和空白对照组(P<0.05)。3组核酸疫苗组抗体诱导水平差异无统计学意义,但Gpd-Tp92融合核酸疫苗组刺激机体引发特异性淋巴细胞增殖能力较之两单基因核酸疫苗组有明显提高。结论 梅毒螺旋体真核表达重组体pcDNA3.1(+)/Gpd-Tp92的成功构建及对新西兰兔产生强特异免疫应答的有效刺激,为梅毒DNA疫苗的研究奠定一定的实验基础。

Construction of fusion bivalent DNA vaccine of Treponema pallidum and its immune activity

Objective To construct the recombinant plasmid of pcDNA3.1(+)/Gpd-Tp92 containing the outer membrane protein Gpd and Tp92 gene of T.pallidum,and observe its immune response in New Zealand white rabbits.Methods Gpd from pcDNA3.1(+)/Gpd was subcloned into the recombinant plasmid pcDNA3.1(+)/Tp92 by linking reactions to construct pcDNA3.1(+)/Gpd-Tp92,and then the recombinant plasmids were transfected into HeLa cells using liposome.After verifying the Gpd-Tp92 antigen fusion genes could be expressed in HeLa cells by immunocytochemistry,three kinds of DNA vaccines comprising pcDNA3.1(+)/Gpd-Tp92,pcDNA3.1(+)/Gpd,pcDNA3.1(+)/Tp92 were administered respectively to three groups of New Zealand white rabbits.The levels of the specific antibody in the sera of rabbits and the cytokine IFN-γin rabbits spleen lymphocyte culture supemant,after stimulating by special proteins, were quantitatively detected by ELISA.The proliferation response of spleen cells was detected by MTT assay.Results Gpd-Tp92 fusion gene constructed in pcDNA3.1(+) vector could express a fusion protein in Hela cells.The significant specific antibody IgG titers were detected and the highest titer was 1:1024 in rabbits of nucleic acid vaccine groups two weeks after three inoculations.The proliferation response of spleen cells and IFN-γlevels induced were significantly higher in rabbits injected with nucleic acid vaccines than those with pcDNA3.1(+) (P<0.05).There was no obvious difference of the levels of specific antibody induced between the double-gene nucleic acid vaccine group and mono-gene nucleic acid vaccine groups, except the more powerful and lasting cellullar immunologic response in double-gene vaccine group.Conelusion All above results establish a solid basis for future study of the biological activities of Gpd and Tp92, and benefit the development of the syphilitic DNA vaccine.