猪δ冠状病毒M蛋白的截短表达及间接ELISA方法的建立  

Truncated Expression of Porcine deltacoronavirus M Protein and Establishment of Indirect ELISA

作　　者：宋代丽 杨富升 陈钰乔 孙凡媛 陈汭 曹三杰 黄小波[1,2,3] SONG Dai-Li;YANG Fu-Sheng;CHEN Yu-Qiao;SUN Fan-Yuan;CHEN Rui;CAO San-Jie;HUANG Xiao-Bo(Research Center of Swine Disease,College of Veterinary Medicine,Sichuan Agricultural University,Wenjiang 611130,China;Sichuan Science-observation Experimental station of Veterinary Drugs and Veterinary Diagnostic Technology,Ministry of Agriculture and Rural affairs,Wenjiang 611130,China;National Teaching and Experiment Center of Animal,Sichuan Agricultural University,Wenjiang 611130,China)

机构地区：[1]四川农业大学动物医学院猪病研究中心,温江611130 [2]农业农村部兽用药物与兽医诊断技术四川科学观测实验站,温江611130 [3]四川农业大学国家级动物类实验教学示范中心,温江611130

出　　处：《农业生物技术学报》2022年第2期402-412,共11页Journal of Agricultural Biotechnology

基　　金：四川省科技创新苗子工程资助项目(2020045);云南省重大科技专项计划(202102AE090039);四川农业大学创新训练计划项目(201810626008)。

摘　　要：猪δ冠状病毒(Porcine deltacoronavirus,PDCoV)是一种新出现的猪(Sus scrofa)肠道致病性冠状病毒,可导致哺乳仔猪严重脱水、呕吐及水样腹泻,在全球范围内广泛分布。为建立一种快速检测PDCoV抗体的间接ELISA方法,本研究利用生物信息学方法分析M蛋白的基因序列特性,设计并扩增PDCoV的M基因序列76~651 bp区域,与表达载体pET-32a构建重组质粒,转化大肠杆菌(Escherichia coil)Transetta(DE3)进行诱导表达,获得截短表达的M蛋白(约35 kD);Western blot实验证明其与猪PDCoV阳性血清有良好的反应原性。基于纯化的重组M截短蛋白构建间接ELISA方法,确定阴阳性判定临界值为0.271。建立的ELISA方法与猪瘟病毒(Classical swine fever virus,CSFV)、猪繁殖与呼吸系统综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪圆环病毒2型(Porcine circovirus type 2,PCV-2)、猪伪狂犬病毒(Pseudorabies virus,PRV)、猪口蹄疫病毒(Foot-and-mouth disease virus,FMDV)、猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)和猪传染性胃肠炎病毒(Swine transmissible gastroenteritis virus,TGEV)等阳性血清无交叉反应,批内和批间变异系数均小于10%;与基于PDCoV S1-CTD蛋白的间接ELISA方法的总符合率高达96%,表明该方法具备良好的特异性、敏感性、重复性及较高的符合率。用建立的ELISA方法检测2012~2018年四川部分地区收集的507份猪血清,结果显示总阳性率为49.11%,初步证明该病已在四川感染严重。本研究基于M截短蛋白构建的间接ELISA方法,为PDCoV疫病诊断和血清流行病学调查提供技术支持,对于PDCoV防控具有一定的意义。Porcine deltacoronavirus(PDCoV)is a new swine(Sus scrofa)enteropathogenic coronavirus,which can cause severe dehydration,vomiting and watery diarrhea in piglets,with worldwide distribution.In order to develop an indirect ELISA method for rapid detection of PDCoV antibody,based on the bioinformatics analysis of PDCoV M gene sequence,the M gene located at 76~651 bp was amplified and inserted into the expression vector pET-32a to construct the recombinant plasmid pET-32a-M.The pET-32a-M was transformed into Escherichia coil Transetta(DE3)and the recombinant M protein was expressed,and a target truncated protein with a size of 35 kD was obtained.Western blot test showed that M protein had good reactivity with porcine PDCoV positive serum.An indirect ELISA method was constructed based on purified recombinant M truncated protein,and the critical value was determined to be 0.271,and had no cross reaction with Classical swine fever virus(CSFV),Porcine reproductive and respiratory syndrome virus(PRRSV),Porcine circovirus type 2(PCV-2),Pseudorabies virus(PRV),Foot-and-mouth disease virus(FMDV),Porcine epidemic diarrhea virus(PEDV),Swine transmissible gastroenteritis virus(TGEV).The coefficient of variation of intra-and inter-batch repeatability tests were less than 10%,and the total coincidence rate with the indirect ELISA based on PDCoV recombinant S1-CTD protein as coating antigen was 96%.The above results showed that the established indirect ELISA method had good specificity,sensitivity,and repeatability.The positive rate of PDCoV antibody was 49.11%in 507 pig serum samples collected in some areas of Sichuan province from 2012 to2018,which preliminarily proved that the disease had been seriously infected in Sichuan.This study established an indirect ELISA method based on recombinant M truncated protein,which could provide technical support for PDCoV epidemic diagnosis and serum epidemiological investigation,and is of certain significance for PDCoV prevention and control.

关 键 词：猪δ冠状病毒(PDCoV) M蛋白 截短表达 间接ELISA 应用 

分 类 号：S855[农业科学—临床兽医学]