登革病毒外膜蛋白DⅢ区融合基因真核表达载体的构建及免疫效果研究

Construction of recombinant eukaryotic plasmids enconding fused envelope domain Ⅲs of dengue virus and its immunogenicity in mice

目的：构建登革病毒外膜蛋白DⅢ区融合基因真核表达载体并分析其免疫原性。方法：利用连接肽将登革病毒1型与2型、3型与4型的外膜蛋白（E蛋白）DⅢ基因片段连接在一起,克隆入真核表达载体pCDNA3.1（＋）,构建DEN1/2型、DEN3/4型EDⅢ融合基因的二价真核表达载体,转染哺乳动物细胞BHK-21,用间接免疫荧光法和免疫印迹法鉴定目的蛋白的表达。将两种二价重组质粒混合形成四价DNA疫苗免疫小鼠,观察其免疫原性及诱导产生中和抗体的能力。结果：二价重组质粒能在BHK-21细胞中表达目的蛋白,四价DNA质粒免疫小鼠,能诱导产生针对登革病毒1~4型的中和抗体,平均中和效价分别为1∶24.7、1∶26.9、1∶13.4、1∶16.0。结论：本研究构建的DNA质粒在小鼠模型中具有良好的免疫原性,为登革多价DNA疫苗的研究提供了可行性。

Objective：To analyze immunogenicity of the fused envelope domain Ⅲs of dengue virus by immunization with DNA vaccine.Methods：Envelope domain Ⅲs of dengue serotypes 1 and 2,3 and 4 were spliced by a linker（Gly-Gly-Ser-Gly-Ser）3 and cloned into eukaryotic vector pCDNA3.1（＋）to generate recombinant bivalent plasmids,then transfected into BHK-21 cells.Expression of the recombinant protein was identified by indirect immunofluorescent assay and Western blot.The tetravalent recombinant plasmids were used to the immunize mice to observe their immunogenicity and ability to induce neutralizing antibodies.Results：The results showed that the recombinant protein could be expressed by BHK-21 cells.Mice immunized with tetravalent plasmids were able to produce neutralizing antibodies against dengue virus type 1 to 4,with neutralizing titers 1∶24.7,1∶26.9,1∶13.4 and 1∶16.0 respectively.Conclusion：It is suggested that the recombinant plasmids are valuable for development of a tetravalent DNA vaccine against dengue virus.