摘要
旨在原核表达犬冠状病毒(CCoV)的刺突(S)蛋白,以此为包被抗原,通过优化条件建立CCoV血清抗体间接ELISA检测方法。首先,将CCoV的S基因序列克隆至原核表达载体pColdⅠ中,在大肠杆菌BL21(DE3)中经IPTG诱导表达。Western-blot验证蛋白大小约为28 ku,纯化后作为包被抗原,通过优化条件建立检测CCoV抗体的间接ELISA,抗原包被最佳质量浓度为4μg/mL;待检血清稀释度为1∶160时D_(450)最高;酶标抗体最佳稀释度为1∶3000;TMB最佳显色时间为20 min;最佳封闭液为50 g/L脱脂奶粉溶液;阴阳性临界值为D_(450)=0.214;与犬瘟热病毒、犬细小病毒、犬腺病毒的阳性血清均不发生交叉反应,表明特异性好;批内变异系数最大值为4.531%,批间变异系数最大值为2.908%,表明重复性好;该方法能检测到640倍稀释的血清,表明敏感性较高。应用该方法对161份临床犬血清进行检测,发现阳性率为98.7%,说明CCoV的感染率较高。本研究为CCoV抗体检测提供了有效工具。
The purpose of this study was to express the spike protein of canine coronavirus(CCoV)in prokaryotic cells,and to establish an indirect ELISA method for the detection of CCoV serum antibody by optimizing the conditions.Firstly,the S gene sequence of CCoV was cloned into the prokaryotic expression vector pCold I and expressed in Escherichia coli BL21(DE3)induced by IPTG.Western-blot showed that the size of the protein was about 28 ku,and the purified protein was used as coating antigen.The indirect ELISA for detecting CCoV antibody was established by optimizing the conditions.The optimal reaction conditions were as follows:the amount of antigen package was 4μg/mL,the sera were diluted with 1∶160,the HRP-IgG was diluted with 1∶3000,and TMB reacted for 20 min;the optimal blocking solution was 50 g/L skimmed milk solution.The positive and negative critical value was D_(450)=0.214.There was no cross reaction with the positive serum of canine distemper virus,canine parvovirus and canine adenovirus,respectively,it showed that the good specificity.The maximum coefficient of variation was 4.531%and 2.908%in intra-and inter-assays,it showed that the good repeatability.The method can detect 640 times diluted serum with high sensitivity.The positive rate was 98.7%,indicating that the infection rate of CCoV was high.This study provides an effective tool for CCoV antibody detection.
作者
曹海旭
胡博
李虹晔
李昀真
代昕宇
张成琪
李双双
张海玲
廉士珍
白雪
CAO Hai-xu;HU Bo;LI Hong-ye;LI Yun-zhen;DAI Xin-yu;ZHANG Cheng-qi;LI Shuang-shuang;ZHANG Hai-ling;LIAN Shi-zhen;BAI Xue(Key Laboratory of Special Animal Epidemic Disease,Ministry of Agriculture and Rural Affairs/Institute of Special Animal and Plant Sciences,Chinese Academy of Agricultural Sciences,Changchun 130112,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2022年第5期564-571,共8页
Chinese Veterinary Science
基金
国家自然科学基金项目(32100408)
吉林省科技发展计划项目(20210202050NC)。
