编码山羊补体C3d与口蹄疫病毒VP1融合蛋白重组质粒的构建及表达

Construction and Eukaryotic Expression of Recombinant Plasmid Encoding Fusion Protein of Goat Complement C3d and Foot-and-Mouth Disease Virus VP1

旨在构建含分子佐剂山羊补体C3d基因的O型口蹄疫病毒VP1基因真核表达质粒。克隆山羊C3d基因,通过linker(G_4S)_2将3拷贝C3d基因串联;克隆羊源O型口蹄疫病毒VP1基因,通过linker(G_4S)_2与3拷贝C3d基因相连,构建重组质粒pUC19-VP1-C3d_3。将VP1-C3d_3融合基因亚克隆入含有分泌表达信号肽tPA序列的pcDNA3.1(+)CMV启动子下游,构建重组真核表达质粒pcDNA3.1-tPA-VP1-C3d_3。在脂质体介导下,将pcDNA3.1-tPA-VP1-C3d_3转染HeLa细胞。间接免疫荧光分析表明,VP1-C3d_3在HeLa细胞中获得了瞬时表达,Western blot分析证实转染的阳性细胞能分泌预期大小(133 kD)的融合蛋白。重组质粒pcDNA3.1-tPA-VP1-C3d_3为研制以羊补体C3d为分子佐剂的口蹄疫新型疫苗奠定了基础。

We constructed a recombinant plasmid encoding VP1 gene of O type foot-and-mouth disease virus fused to a molecular adjuvant, goat complement C3d gene. The goat C3d gene was cloned and three copies were tandem-linked with the linker （G4S）2 sequence. VP1 gene of O type foot-and-mouth disease virus was linked to three tandem repeats of C3d through the linker sequence and cloned into pUC19 to obtain the recombinant plasmid pUC19-VP1-C3d3. The VP1-C3d3 fusion gene was then subcloned into the eukaryotic vector pcDNA3.1（＋） that had been modified to contain the tissue plasminogen activator （tPA） leader sequence to obtain pcDNA3.1-tPA-VP1-C3d3. HeLa cells were transfected with pcDNA3.1-tPA-VP1-C3d3 by Lipofectamine^TM 2000. Indirect immunofluorescent assay and Western blot assay showed that VP1-C3d3 fusion gene was successfully expressed in HeLa cells. The fusion protein with the expected size 133 kD could be secreted outside the cells. This study laid a good foundation to further research on the novel vaccine against foot-and-mouth disease virus by using goat C3d as a molecular adjuvant to enhance the immunogenicity of VP1.