中国动物传染病学报2024，Vol.32Issue(6)：120-127,8.
牛冠状病毒N蛋白的原核表达及其单克隆抗体的制备
Prokaryotic Expression of N Protein of Bovine Coronavirus and Preparation of Monoclonal Antibodies
摘要
Abstract
In order to express the N protein of bovine coronavirus(BCoV)and prepare anti-N protein monoclonal antibodies(mAbs),the N gene was cloned into the prokaryotic vector pCold-GST for expression in E.coli BL21(DE3).The results showed that the recombinant N protein was mainly released as soluble form and had a molecular weight of 77 kDa as visualized in SDS-PAGE.The purity of the recombinant N protein reached 90%after affinity purification.In addition,Western blotting results confirmed that the recombinant N protein had good reactivity with the serum collected from BCoV-immunized rabbits.Then,the purified N protein was used to immunize to BALB/c mice to generate mAbs.Finally,three mAbs 1D11,5C2 and 2B11were obtained by screening hybrodomas.The titers of these three mAbs in the cell culture supernatants reached to 1:320.All mAbs belonged to IgG1subclass.The results of Western blot and IFA demonstrated there were specific interactions between these mAbs and N protein from prokaryotic expression.The availability of mAbs provided materials for further research on the structure and function of N protein and rapid diagnosis of BCoV infection.关键词
牛冠状病毒/原核表达/N蛋白/单克隆抗体Key words
Bovine coronavirus/prokaryotic expression/N protein/monoclonal antibody分类
农业科技引用本文复制引用
梁彤,常继涛,曹存,王芳,姜志刚,于德斌,徐晓静,崔久辉,贾斌,尹鑫..牛冠状病毒N蛋白的原核表达及其单克隆抗体的制备[J].中国动物传染病学报,2024,32(6):120-127,8.基金项目
内蒙古自治区科技重大专项(2020ZD0006,2021ZD0013) （2020ZD0006,2021ZD0013）
