猪δ冠状病毒结构蛋白的表达及其多克隆抗体的制备  

Construction 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[2,3,4] 李彬 主性[1] ZHANG Baotai;XIAO Li;ZHONG Chunyan;ZHOU Jinzhu;HUANG Jin;YUAN Xuesong;CAI Xuhang;LI Siyuan;GUO Rongli;LI Jizong;LI Bin;ZHU Xing(College of Animal Science,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;Institute of Life Sciences,School of Food and Biological Engineering,Jiangsu University,Zhenjiang 212013,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Biological Engineering Department,Southwest Guizhou Vocational and Technical College for Nationalities,Xingyi 562400,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/农业部兽用生物制品工程技术重点实验室,江苏南京210014 [3]江苏大学生命科学学院,江苏镇江212013 [4]南京农业大学动物医学院,江苏南京210095 [5]黔南民族职业技术学院生物工程系,贵州兴义562400 [6]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《畜牧与兽医》2023年第12期68-74,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：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 complete 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 wereconstructed.The plasmids were identified by sequencing and were transfected to HEK293T cells,and the expressions of PDCOV S,E,Mand 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 pCAGGSPDCoV-S,pCAGGS-PDCoV-E,pCAGGS-PDCoV-M,pCAGGS-PDCoV-N were successfully constructed and expressed in HEK293Tcells.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 preparedby 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.

关 键 词：PDCoV S、E、M、N基因 真核表达 抗原性 

分 类 号：S852[农业科学—基础兽医学]