基于猪流行性腹泻病毒S1蛋白间接ELISA抗体检测方法的建立及初步应用  

Establishment and preliminary application of an indirect ELISA based on S1 protein of porcine epidemic diarrhea virus

作　　者：罗烨 罗灵芝 丁彦彬 赵墩[1,2] 周小飞 郑金 李润成 葛猛[1] 余兴龙[1] LUO Ye;LUO Ling-zhi;DING Yan-bin;ZHAO Dun;ZHOU Xiao-fei;ZHENG Jin;LI Run-cheng;GE Meng;YU Xing-long(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China;Hunan Kangbaote Biotechnology CO.,LTD.,Changsha 410128,China)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]湖南康保特生物科技有限公司,湖南长沙410128

出　　处：《中国兽医科学》2020年第11期1341-1347,共7页Chinese Veterinary Science

基　　金：国家自然科学基金面上项目(31672539);湖南省重点领域研发计划项目(2019NK2171)。

摘　　要：根据猪流行性腹泻病毒(PEDV)HuN株S1蛋白的基因序列(GenBank:JQ517274)构建哺乳动物细胞重组表达质粒pIRES-S1。利用电转法将pIRES-S1转染至ST细胞,经过G418加压筛选、Western-blot检测、悬浮驯化后,得到能表达S1蛋白的ST稳转细胞池。Western-blot结果显示,S1蛋白具有良好的反应原性。以S1蛋白作为包被抗原初步建立间接ELISA方法,并对PEDV临床血清和进口种猪PEDV阴性血清进行检测,以血清中和试验为标准,结果显示,该ELISA方法敏感性为96.3%,特异性为97.7%,与血清中和试验具有很高的一致性(kappa值=0.882,P<0.05)。应用ELISA方法对PEDV返饲猪跟踪血清检测,结果显示,该方法能准确地评估感染猪体内PEDV IgG抗体水平消长规律。上述结果表明,本研究建立的基于S1蛋白的ELISA方法具有高敏感性和高特异性,可用于临床猪血清中PEDV抗体的检测,为猪群PEDV的免疫评估提供有效依据。The recombinant expression plasmid pIRES-S1 was constructed according to the gene sequence of PEDV S1 in NCBI(GenBank:JQ517274).The plasmid pIRES-S1 was transfected into ST cells by electrotransfer.After G418 pressurization screening,western-blot detection and suspension domestication,a stable transduction cell pool expressing S1 protein was obtained.The results of Western-blot showed that S1 protein have good reactivity.An indirect ELISA was established by using S1 protein as coating antigen,and the ELISA was used to detect PEDV clinical serum and PEDV negative serum of imported breeder pigs.Take the serum neutralization test as the standard,the results showed that the sensitivity of the ELISA was 96.3%and the specificity was 97.7%.It was significantly consistent with the serum neutralization test(kappa value=0.882,P<0.05).The ELISA was used to detect the tracking serum of PEDV back-feeding pigs.The results showed that it could accurately evaluate the growth and decline of PEDV IgG antibody level in infected pigs.Our results suggested that the ELISA based on S1 protein established in this study has high sensitivity and specificity.It could be used to detect PEDV antibody in clinical serum samples and provide an effective basis for immune evaluation of PEDV in pigs.

关 键 词：猪流行性腹泻病毒 S1蛋白 ST细胞 间接ELISA 

分 类 号：S852.659.6[农业科学—基础兽医学]