牛冠状病毒N基因的原核表达和多克隆抗体的制备  

Prokaryotic Expression of Bovine Coronavirus N Gene and Preparation of Polyclonal Antibody

作　　者：刘文锴 曹兴旺 汪萍[2,3,4] 金映红 薛晶[2,3,4] 李月 梁纤纤[5] 李晓卓 韩翔舒 郑启铭 蒋松 夏俊 LIU Wen-kai;CAO Xing-wang;WANG Ping;JIN Ying-hong;XUE Jing;LI Yue;LIANG Xian-xian;LI Xiao-zhuo;HAN Xiang-shu;ZHENG Qi-ming;JIANG Song;XIA Jun(College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang,832061,China;Institute of Veterinary Medicine,Xinjiang Academy of Animal Husbandry Sciences,Urumqi,Xinjiang,830000,China;The Key Laboratory for Prevention and Control of Herbivore Diseases of the Ministry of Agriculture and Rural Affairs,Urumqi,Xinjiang,830000,China;Xinjiang Key Laboratory of Animal Disease Research,Urumqi,Xinjiang,830000,China;College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi,Xinjiang,830000,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832061 [2]新疆畜牧科学院兽医研究所,新疆乌鲁木齐830000 [3]农业农村部草食动物疫病防控重点实验室,新疆乌鲁木齐830000 [4]新疆动物疫病研究重点实验室,新疆乌鲁木齐830000 [5]新疆农业大学动物医学学院,新疆乌鲁木齐830000

出　　处：《动物医学进展》2025年第3期23-27,共5页Progress In Veterinary Medicine

基　　金：天山英才-青年科技拔尖人才项目(2022TSYCCX0046);新疆重大专项(2023A02007);国家级大学生创新创业项目(202310759012)。

摘　　要：为制备牛冠状病毒N蛋白多克隆抗体,建立简单快捷的牛冠状病毒检测方法。根据牛冠状病毒N基因序列设计引物,将N基因重组至PET-30a载体上,经PCR及测序鉴定重组载体构建成功;将构建的PET-30a-N重组质粒转入大肠埃希氏菌DH5-α和Rosetta(DE3)感受态细胞,进行扩增和诱导表达,后经超声破碎后获取N蛋白并进行蛋白纯化;最后将纯化后的N蛋白与弗氏佐剂乳化后免疫新西兰白兔,共制得35 mL抗N蛋白多克隆抗体血清,经Western blot和IFA鉴定,抗N蛋白多克隆抗体有良好的特异性,试验制备的抗N蛋白牛冠状病毒多克隆抗体可用于牛冠状病毒的检测。In order to prepare polyclonal antibody against bovine coronavirus N protein,a simple and rapid detection method for bovine coronavirus was established.A pairs of primers were designed according to the bovine coronavirus N gene sequences,and the N gene was recombined into PET-30a vector.The recombinant vector was successfully constructed by PCR and sequencing.The constructed PET-30a-N recombinant plasmid was transferred into Escherichia coli DH5-αand Rosetta(DE3)receptor cells for amplification and induced expression,and the N protein was obtained and purified after ultrasonic crushing.Finally,the purified N protein was emulsified with Freund’s adjuvant and used to immunize New Zealand white rabbits,and a total of 35mL anti-N protein polyclonal antibody serum was prepared.Western blot and IFA identification showed that the anti-N protein polyclonal antibody had good specificity.The prepared polyclonal antibody against N protein of bovine coronavirus can be used for the detection of bovine coronavirus.

关 键 词：牛冠状病毒 N蛋白 多克隆抗体 原核表达 

分 类 号：S852.65[农业科学—基础兽医学]