猪流行性腹泻病毒N-E.coli OmpA融合基因构建、表达及其免疫原性评价

Construction,Expression of Fusion Gene PEDV N-E.coli OmpA and Immunogenicity Evaluation of Its Fusion Protien

为获得免疫原性更强的猪流行性腹泻病毒(PEDV)结构蛋白,试验将PEDV N蛋白基因和大肠杆菌(E.coli)OmpA蛋白基因进行融合,采用柔性linker联接,得到OmpA-linker-PEDV N融合基因,并克隆至pCold I质粒,构建重组质粒pCold I-OmpA-linker-PEDV N。重组质粒转入BL21感受态细胞,SDS-PAGE分析重组质粒的表达,并对其编码的融合蛋白进行二级和三级结构预测。Western-blot和ELISA分析融合蛋白的免疫原性。结果表明:构建了融合基因表达质粒pCold I-OmpA-linker-PEDV N,表达了约88 ku的融合蛋白;融合蛋白的二级结构未发生明显变化,三级结构预测可折叠为正确的空间构像。该融合蛋白同时具有PEDV N蛋白和OmpA蛋白的免疫原性,并且其免疫原性显著高于单一PEDV N蛋白。说明试验获得了免疫原性较好的融合基因表达蛋白,可以为PEDV N蛋白功能研究、特异性抗体制备以及PEDV亚单位疫苗的研制提供材料和研究基础。

In order to obtain a more immunogenic porcine epidemic diarrhea virus(PEDV)structural protein,the PEDV N protein gene and E.coli OmpA protein gene were fused by a flexible linker,and obtain fusion gene OmpA-linker-PEDV N,which was cloned into pCold I expression plasmid to construct the recombinant plasmid pCold I-OmpA-linker-PEDV N.The recombinant plasmid was transformed into Escherichia coli BL21,SDS-PAGE analyzed the expression of the recombinant plasmid,and predicts the secondary and tertiary structure of the fusion protein.The expressed fusion protein was purified and its immunogenicity was analyzed by Western-blot and indirect ELISA.The results showed that the pCold I-OmpA-linker-PEDV N fusion gene plasmid was obtained,and approximate 88 ku fusion protein was expressed.The secondary structure of the fusion protein did not change significantly,and the tertiary structure predicts that it can be folded into the correct spatial conformation.The fusion protein has both the immunogenicity of PEDV N protein and OmpA protein;and its immunogenicity is significantly higher than that of PEDV N protein.These results indicate that the fusion gene expression protein with better immunogenicity was obtained in the experiment.The fusion protein can provide materials and research basis for the study of PEDV N protein function,preparation of specific antibodies,and the development of PEDV subunit vaccines.