基于重组EP153R蛋白的检测非洲猪瘟病毒抗体的间接ELISA方法的建立  

Development of Indirect ELISA for Detection of African Swine Fever Virus Antibodies Based on Recombinant EP153R Protein

作　　者：程佳[1] 李遥 刘英楠 钟秋萍 史馨瑾 魏常青 谢振华 廖欣欣 宋影影 陈鸿军[2,3] CHENG Jia;LI Yao;LIU Yingnan;ZHONG Qiuping;SHI Xinjin;WEI Changqing;XIE Zhenhua;LIAO Xinxin;SONG Yingying;CHEN Hongjun(College of Veterinary Medicine,Shanxi Agricultural University,Taiyuan 030801,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Biosafety Research Center,CASS,Shanghai 200241,China)

机构地区：[1]山西农业大学动物医学学院,山西030801 [2]中国农业科学院上海兽医研究所,上海200241 [3]中国农业科学院生物安全研究中心,上海200241

出　　处：《中国动物传染病学报》2022年第2期74-83,共10页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金面上项目(32170161);国家自然基金联合基金项目重点支持项目-区域创新发展联合基金项目(U19A2039)。

摘　　要：为探究非洲猪瘟病毒(ASFV)EP153R蛋白的功能和建立ASFV抗体间接ELISA方法,本试验构建了EP153R蛋白杆状病毒表达系统,采用Western blot与IFA鉴定rEP153R蛋白的表达和免疫原性。结果显示,杆状病毒能高效表达rEP153R蛋白且可被小鼠多克隆抗体识别,具有良好的免疫原性。以此重组蛋白为抗原进行间接ELISA方法的建立,经方阵ELISA确定当抗原包被量2.5μg/mL,ASFV阳性血清稀释比1∶1000时为最佳反应条件;以优化后的条件确定了抗体阳性临界值为0.259;特异性试验结果表明,该方法仅与ASFV阳性血清发生反应,具有较强的特异性;重复性试验结果显示变异率小于10%,重复性良好。利用该方法对46份猪血清进行检测,与商品化试剂盒检测结果对比显示,符合率为91.3%,能较好的区分ASFV阴性血清与阳性血清。本研究的结果为后续EP153R功能研究和ASFV临床血清学诊断奠定了基础。To explore the function of African swine fever virus(ASFV)EP153R protein and develop an indirect ELISA for detecting antibodies against ASFV,the recombinant EP153R was produced using the baculovirus expression system.The immunogenicity of the recombinant rEP153R protein was determined in Western blot and IFA.The results showed that the recombinant EP153R protein was expressed in baculovirus and recognized by mouse polyclonal antibodies.Subsequently,an indirect ELISA method was developed and optimized using the recombinant EP153R protein at 2.5μg/mL and positive ASFV serum at 1:1000 dilution.The cutoff value for positive antiserum was determined to be 0.259.This indirect ELISA method reacted with ASFV positive serum samples only,indicating its good specificity.This method also had good repeatability as the variability among tests was less than 10%.Then this method was used to detect 46 pig serum samples and the test results were compared with those of a commercial kit.The results showed that this indirect ELISA method had an agreement of 91.3%with the commercial kit and accurately detected ASFV negative and positive serum samples.In summary,the results of this study laid a strong foundation for future research on EP153R function and serological diagnosis of ASFV.

关 键 词：非洲猪瘟病毒 杆状病毒 EP153R ELISA 

分 类 号：S852.651[农业科学—基础兽医学]