畜牧与兽医2025，Vol.57Issue(8)：69-77,9.
牛冠状病毒S蛋白生物信息学分析及其受体结合域的多克隆抗体制备
Bioinformatic analysis of bovine coronavirus S protein and preparation of polyclonal antibody against its receptor-binding domains
摘要
Abstract
This study was to predict and analyze the structure and potential function of bovine coronaviruses(BCoV)S protein,and to clone and express the protein and prepare polyclonal antibodies,so as to provide the basis for vaccine research and laboratory diagnosis.Bioinformatics software was used to predict and analyze the biological characteristics of S protein,its receptor binding domain(RBD)gene was cloned and linked to prokaryotic expression vector pET-30a(+).Recombinant plasmids were constructed.BL21 receptor cells were transformed and their expression was induced by IPTG.The expressed protein was purified by nickel column and was immunized mice to pre-pare polyclonal antibodies which were then detected.The results were that S protein was composed of 1 364 aa,with a relative molecular weight of 150.81 kDa,the isoelectric point at 5.51,an instability index of 33.22,and a fat solubility index of 87.22.The amino acids 1-1 307 of the protein were in an extracellular region,1 308-1 330 aa were in a transmembrane region,1 331-1 363 aa were in an intracellu-lar region,1-14 aa were signalpeptides,and 310-612 aa were RBD.There were 36 potential B-cell epitopes,12 of which were contained in RBD,with α-helix accounting for 27.95％,β-fold accounting for 23.04％,and random curling accounting for 49.01％,respectively.The analysis results of S protein tertiary structure were basically consistent with the prediction results of the secondary structure.The size of RBD of S protein successfully cloned here was consistent with the expectation,and the vector was successfully constructed by double enzyme diges-tion and sequenced after being connected to the vector pET-30a(+).After the successful expression of the protein was verified by SDS-PAGE,the purified protein was immunized on mice and serum samples were isolated.The polyclonal antibody was verified by indirect immu-nofluorescence to recognize the corresponding antigen,which proved that the recombinant protein had good immunogenicity.The structure and function of BCoV S protein were analyzed and predicted successfully,and the polyclonal antibodies of the S protein receptor binding domain were obtained,which laid the foundation for subsequent studies.关键词
牛冠状病毒/S蛋白/受体结合域/生物特性/多克隆抗体Key words
BCoV/S protein/receptor binding domain/biological characteristics/polyclonal antibody分类
农业科技引用本文复制引用
韩翔舒,平继辉,蒋松,夏俊,宫震,刘淑悦,范昊宇,付修龙,李晓卓,梁纤纤,郑启铭,李月..牛冠状病毒S蛋白生物信息学分析及其受体结合域的多克隆抗体制备[J].畜牧与兽医,2025,57(8):69-77,9.基金项目
新疆维吾尔自治区天山英才青年科技拔尖人才项目(2022196128) （2022196128）
新疆维吾尔自治区重大科技专项项目(2022273334) （2022273334）
新疆维吾尔自治区肉牛产业技术体系项目(XJARS-10) （XJARS-10）
新疆畜禽疫病防控体系质量提升工程项目(2023A02007) （2023A02007）
