猪δ冠状病毒结构蛋白的表达及其多克隆抗体的制备  

Expression of structural protein of porcine deltacoronavirus and preparation of its polyclonal antibody

作　　者：张宝太 肖丽 钟纯燕 周金柱 黄金 袁雪松 蔡旭航 李思远 郭荣利 李基棕[2,5,6] 李彬 主性[1] ZHANG Baotai;XIAO Li;ZHONG Chunyan;ZHOU Jinzhu;HUANG Jin;YUAN Xuesong;CAI xuhang;LI iyuan;CUO IZONG;LI Bin;ZH Xing(College of Animal Sciences,Guizhou University,Guiyang 550025,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture and Rural Affairs,Nanjing 210014,China;Biological Engineering Department,Southwest Guizhou Vocational and Technical College for Nationalities,Xingyi 562400,China;School of Life Sciences,School of Food and Biological Engineering,Jiangsu University,Zhenjiang 212013,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]江苏省农业科学院兽医研究所/农业农村部兽用生物制品工程技术重点实验室,江苏南京210014 [3]黔南民族职业技术学院生物工程系,贵州兴义5624007 [4]西北农林科技大学动物医学院,陕西杨凌712100 [5]江苏大学生命科学学院,食品与生物工程学院,江苏镇江212013 [6]南京农业大学动物医学院,江苏南京210095

出　　处：《畜牧与兽医》2023年第8期86-92,共7页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划(2022YFD1800803);江苏省自然科学基金(BK20221432)。

摘　　要：旨在获得猪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,评价免疫原性。结果表明,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4种重组蛋白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。本研究制备的多克隆抗体具有较强的免疫原性,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 complete S,E,M and N genes of the PDCoV CZ2020 strain were amplified by RT-PCR and cloned to a pCAGCS eukaryotic expression vectors;and eucaryotic expression plasmids pCAGGS-PDCoV-S,pCAGCS-PDCoV-E,pCAGGS-PDCoV-M and pCAGGS-PDCoV-N were constructed.After sequencing identification,the plasmids were transfected to HEK293T cells,and the expression of PDCOV S,E,M and N proteins were detected by indirect immunofluorescence test(IFA)and Western blot(WB).Then,BALB/c mice were prepared for subcutaneous injection of the proteins,and recombinant proteins polyclonal antibodies were produced in the immunized mice.Finally,the titers of the antibodies were measured by WB and IFA,and their immunogenicity was evaluated.The results showed that the recombinant plasmids pCACCS-PDCoV-S,pCAGGS-PDCoV-E,pCAGCS-PDCoV-M,and 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 pCACGS-PDCoVS,pCACCS-PDCoV-E,pCAGGS-PDCoV-M,and pCAGGS-PDCoV-N was as high as 1:10000.It suggested that the vaccines prepared using recombinant proteins induced mice into producing higher levels of antibodies.The results of this study indicated that the pCAGCS-PDCoV-S,pCAGGS-PDCoV-E,pCAGGS-PDCoV-M and pCAGCS-PDCoV-N eucaryotic expression vectors were successfully constructed here.The polyclonal antibodies prepared were highly immunogenic,which provided a basis for development of diagnostic kits and new vaccines.

关 键 词：PDCoV 真核表达 抗原性分析 

分 类 号：S852.65[农业科学—基础兽医学]