猪δ冠状病毒RBD的可溶性表达、纯化及小鼠抗血清制备  

Soluble expression and purification of PDCoV RBD protein in mammalian cells and preparation of mouse antiserum

作　　者：崔春梅 张爽 张国庆 蒋嵘[2] 高子函 郝鹏飞 李昌 金鑫 李乐天[2,3] 金宁一 CUI Chunmei;ZHANG Shuang;ZHANG Guoqing;JIANG Rong;GAO Zihan;HAO Pengfei;LI Chang;IN Xin;LI Letian;JIN Ningyi(College of Agriculture,Yanbian University,Yanji,Jilin 133002,China;Research Unit of Key Technologies for Prevention and Control of Virus Zoonoses,Chinese Academy of Medical Sciences,Changchun 130122,China;Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130122,China)

机构地区：[1]延边大学农学院,吉林延吉136200 [2]中国医学科学院人兽共患病毒病防控关键技术研究创新单元,吉林长春130122 [3]中国农业科学院长春兽医研究所,吉林长春130122

出　　处：《中国兽医学报》2024年第5期953-958,共6页Chinese Journal of Veterinary Science

基　　金：中国医学科学院医学与健康科技创新工程资助项目(2020-12M-5-001);国家重点研发计划项目子课题资助项目(2021YFD1801103)。

摘　　要：为获得猪δ冠状病毒(PDCoV)S蛋白受体结合区(RBD)的可溶性蛋白及特异性多克隆抗体血清,本研究将PDCoV流行株S蛋白(GenBank:QAA06990.1)的RBD序列添加信号肽、纯化标签与酶切位点,命名为PDCoV-RBD(简称PDR),对其进行密码子优化后合成并克隆至pcDNA3.1(+)真核表达载体,构建重组质粒pcDNA3.1-PDR。将重组质粒转染至Expi293F细胞进行表达,并利用SDS-PAGE与Western blot进行鉴定;通过Strep-Tactin XT亲和层析纯化目的蛋白,薄层扫描分析蛋白纯度,并以纯化后的PDR蛋白免疫小鼠,制备抗PDR蛋白的特异性多克隆抗体血清,通过间接ELISA测定效价,PDCoV病毒感染细胞后,通过Western blot检测S蛋白与血清的特异性结合效果。结果显示,PDR在Expi293F细胞中获得可溶性表达,纯化得到的目的蛋白大小正确,纯度大于98%;用其免疫小鼠制备的抗血清能特异性结合病毒中天然的S蛋白。本研究获得的PDR蛋白及其小鼠抗血清可为PDCoV的诊断检测与疫苗研制提供必要的物质基础。Soluble protein and specific polyclonal antibody serum against receptor binding domain(RBD)of the porcine delta coronavirus(PDCoV)S protein were obtained.The sequence of the S protein(GenBank:QAA06990.1)of the PDCoV epidemic strain was modified by adding the sequences of a signal peptide,purification tag,and enzyme cleavage site,then it was named PDCoV-RBD(abbreviated as PDR).The PDR sequence was codon-optimized,synthesized and cloned into the pcDNA3.1(+)eukaryotic expression vector to construct the recombinant plasmid pcDNA3.1-PDR.The recombinant plasmid was transfected into Expi293F cells for expression,and the expressed proteins were identified by SDS-PAGE and Western blot.Strep-Tactin XT affinity chromatography was used to purify the target protein,and thin-layer scanning was used to analyze the purity of the protein.The specific polyclonal antibody serum against PDR protein was prepared by immunizing mice with the PDR protein,and the titer was determined by indirect ELISA.The effect of specific binding of PDCoV S protein to serum was detected by Western blot after infecting cells with the PDCoV virus.The results showed that PDR was expressed soluble in Expi293F cells,and the size of the purified target protein was correct,and its purity exceeded 98%.The antiserum prepared can specifically bind to the target protein.These studies laid a solid foundation for serological detection,epidemiological investigation,and subunit vaccine development of PDCoV.

关 键 词：猪δ冠状病毒 受体结合区蛋白 表达 纯化 抗血清制备 

分 类 号：Q78[生物学—分子生物学] S852.65[农业科学—基础兽医学]