为了获得猪δ冠状病毒(PDCoV)结构蛋白并评价其免疫原性,通过RT-PCR技术,扩增PDCoV CZ2020株中完整的S、E、M和N基因,克隆至 pCAGGS 真核表达载体,构建 pCAGGS-PDCoV-S、pCAGGS-PDCoV-E、pCAGGS-PDCoV-M、pCAGGS-PDCoV-N 真核表达质粒,经测序鉴定后转染HEK293T细胞,采用间接免疫荧光试验(IFA)和Western blot(WB)方法检测PDCoV的S、E、M、N蛋白表达.将4种蛋白分别皮下注射BALB/c小鼠,制备重组蛋白多克隆抗体,并用WB法和IFA法测定抗体效价,评价免疫反应性.结果:重组质粒pCAGGS-PDCoV-S、pCAGGS-PDCoV-E、pCAGGS-PDCoV-M、pCAGGS-PDCoV-N能够在HEK293T细胞内正常表达,并与PDCoV阳性猪血清特异性结合;制备的pCAGGS-PDCoV-S、pCAGGS-PDCoV-E、pCAGGS-PDCoV-M 和 pCAGGS-PDCoV-N 的多克隆抗体效价可达 1∶10 000 以上,表明重组蛋白可诱导小鼠产生高水平抗体.结果表明pCAGGS-PDCoV-S、pCAGGS-PDCoV-E、pCAGGS-PDCoV-M和pCAGGS-PDCoV-N真核表达质粒成功构建,试验制备的多克隆抗体具有较强的免疫反应性,能够为研制诊断试剂盒和新型疫苗提供基础.
In order to obtain the structural protein of the porcine deltacoronavirus(PDCoV)and to evaluate its immunogenicity,the com-plete S,E,M and N genes of the PDCoV CZ2020 strain were amplified by RT-PCR,and cloned to pCAGGS eukaryotic expression vectors;and then eucaryotic expression plasmids pCAGGS-PDCoV-S,pCAGGS-PDCoV-E,pCAGGS-PDCoV-M and pCAGGS-PDCoV-N were constructed.The plasmids were identified by sequencing and were transfected to HEK293T cells,and the expressions of PDCOV S,E,M and N proteins were detected by indirect immunofluorescence test(IFA)and Western blot(WB).BALB/c mice were prepared for subcuta-neous injection of the proteins,and recombinant proteins polyclonal antibodies were produced in immunized mice.Next,the titers of the anti-bodies were measured by WB and IFA,and their immunogenicity was evaluated.The results showed that the recombinant plasmids pCAGGS-PDCoV-S,pCAGGS-PDCoV-E,pCAGGS-PDCoV-M,pCAGGS-PDCoV-N were successfully constructed and expressed in HEK293T cells.The recombinant proteins were specifically bounded to anti-pig PDCoV serum.The polyclonal potency of pCAGGS-PDCoV-S,pCAGGS-PDCoV-E,pCAGGS-PDCoV-M,pCAGGS-PDCoV-N was as high as 1∶10 000.These results indicated that vaccines prepared by recombinant proteins would be able to induce mice to produce higher levels of antibodies.This study suggested that pCAGGS-PDCoV-S,pCAGGS-PDCoV-E,pCAGGS-PDCoV-M and pCAGGS-PDCoV-N eucaryotic expression vectors were successfully constructed.The poly-clonal antibodies prepared were highly immunogenic and could serve as a basis for development of diagnostic kits and new vaccines.