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 expression and purification of PDCoV RBD protein in mammalian cells and preparation of mouse antiserum
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 se-quences 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 ex-pressed proteins were identified by SDS-PAGE and Western blot.Strep-Tactin XT affinity chroma-tography was used to purify the target protein,and thin-layer scanning was used to analyze the pu-rity 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 sero-logical detection,epidemiological investigation,and subunit vaccine development of PDCoV.
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