摘要
为建立猪急性腹泻综合征冠状病毒(SADS-CoV)S1蛋白抗体的间接ELISA检测方法,本研究通过构建重组真核分泌型表达质粒p CAGGS-S1-His并转染HEK293F悬浮细胞进行可溶性表达S1蛋白。转染重组质粒5 d后的细胞上清利用HisTrapTMExcel亲和层析柱纯化,浓缩后的蛋白经SDS-PAGE、western blot鉴定显示获得了分子量约为130 ku且大于理论大小的重组S1蛋白,表明该蛋白为分泌表达且具有良好的翻译后修饰。利用获得的重组S1蛋白作为包被抗原,通过优化反应条件,初步建立了SADS-CoV S1蛋白抗体的间接ELISA检测方法。该方法与猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪德尔塔冠状病毒(PDCoV)、猪轮状病毒(PoRV)和猪圆环病毒2型(PCV2)阳性血清均无交叉反应,仅与SADS-CoV阳性血清反应,表明该方法特异性较强;该方法检测SADS-CoV阳性血清,当稀释至1:3 200时仍为阳性,表明该方法具有较高的敏感性;对4份阳性血清的批内和批间重复性试验的变异系数均小于10%,表明该方法具有良好的重复性和稳定性;利用该方法和间接免疫荧光试验(IFA)对50份临床样品检测,结果显示该间接ELISA检测到9份阳性样品,41份阴性样品;IFA检出5份阳性样品,45份阴性样品,二者的总符合率为92%。综上表明,本研究建立的间接ELISA检测方法能够特异、灵敏地检测SADS-CoV S1蛋白抗体,可以用于SADS-CoV灭活疫苗的免疫效果评价以及该病原的血清学流行病学调查和免疫水平监测,为SADS-CoV所致疫病的鉴别诊断和防控奠定基础。
To establish an indirect ELISA method for detection of antibodies to the S1 protein of swine acute diarrhea syndrome coronavirus(SADS-CoV),the present study constructed a recombinant eukaryotic secretory expression plasmid,referred as pCAGGS-S1-His.Subsequently,HEK293F suspension cells were transfected with the recombinant plasmid for expression and purification of the soluble S1 protein.Five days post transfection,the supernatant of cells was purified by HisTrapTM Excel affinity chromatography column,and the concentrated protein was identified by SDS-PAGE and western blot.The result showed that the recombinant S1 protein with a molecular weight of about 130ku was observed,which was larger than the theoretical size,indicating that the protein could be secreted and expressed and had a good post-translational modification.Using the purified recombinant S1 protein as a coating antigen,an indirect ELISA method was established for the detection of antibody to porcine acute diarrhea syndrome coronavirus S1 protein.The method is combined with porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),porcine deltacoronavirus(PDCoV),porcine rotavirus(PoRV)and porcine circovirus(PCV2)showed no cross reaction,and only reacted with SADS-CoV positive sera,which indicated that the specificity of this method was good.The detection of antibodies against the immune sera against SADS-CoV was still tested positive when the antibody was diluted to 1:3200,which indicated that the method had a high degree of sensitivity.The coefficients of variation for the intra-batch and inter-batch tests were less than 10%,which shows that this method has good reproducibility and stability.50 clinical samples were tested using this method and an indirect immunofluorescence assay(IFA),which showed a 92%compliance rate between the two methords.In conclusion,the indirect ELISA method established in this study can effectively and specifically detect antibodies against SADS-CoV S1 protein,which can be valuable for evaluating antibody responses against inactivated SADS-CoV vaccines,conducting epidemiological investigation and monitoring immunization effect.It also provides a solid foundation for the differential diagnosis,prevention and control of disease caused by SADS-CoV.
作者
杨小曼
时洪艳
张燎原
张鑫
张记宇
刘大凯
冯廷帅
曾苗苗
陈建飞
石达
冯力
YANG Xiao-man;SHI Hong-yan;ZHANG Liao-yuan;ZHANG Xin;ZHANG Ji-yu;LIU Da-kai;FENG Ting-shuai;ZENG Miao-miao;CHEN Jian-fei;SHI Da;FENG Li(State Key Laboratory for Animal Diease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2024年第6期608-613,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
国家重点研发计划(2021YFD1801105)
广西兽医生物技术重点实验室开放基金(22-035-32-B-01)
中国农业科学院基本科研业务费(1610302022015)。
作者简介
杨小曼(1997-),女,河南洛阳人,硕士研究生,主要从事猪肠道冠状病毒研究;通信作者:石达,E-mail:shida@caas.cn;通信作者:冯力,E-mail:fengli@caas.cn。
