猪丁型冠状病毒S1基因的克隆、原核表达及多克隆抗体制备

摘要

本研究旨在克隆猪丁型冠状病毒(PDCoV)S1基因(1—1 566 bp),体外表达 S1基因抗原表位预测区Sa(106—1 290 bp)蛋白并制备抗该蛋白质的多克隆抗体.运用生物信息学软件分析 S1核苷酸和编码氨基酸序列特征,将 S1基因抗原表位预测区Sa克隆至原核表达载体pET22b(+),构建重组表达载体pET22b-Sa,转化Rosetta(DE3)菌株,IPTG诱导表达、超滤柱浓缩纯化重组蛋白、Ni柱亲和层析,Western blot检测Sa蛋白的活性,将Sa蛋白免疫新西兰兔制备多克隆抗体.结果表明,CHN-2015毒株 S1基因开放型阅读框大小为1 566 bp(GenBank No.KY398009),编码522个氨基酸,是具有亲水性的非跨膜蛋白,存在15个潜在的B细胞抗原表位;原核表达Sa重组蛋白,该蛋白质在37 ℃下用终浓度为0.8 mmol·L －1IPTG诱导5 h后,蛋白质主要以可溶性形式存在,免疫印迹显示表达的Sa蛋白在上清与包涵体中都有良好的反应性;用纯化的Sa蛋白四次免疫家兔,获得的兔抗Sa蛋白抗体效价可达1:10 240.本研究克隆了 S1基因,原核表达了Sa蛋白和制备了高效价的兔抗Sa蛋白抗体,具有良好的免疫原性,为后续深入开展S蛋白的功能研究奠定了基础.%In order to obtain polyclonal antibody against porcine deltacoronavirus(PDCoV)spike-1(S1)protein,we cloned the gene(1-1 566 bp),and then expressed the Sa protein(106-1 290 bp)of S1 gene epitope prediction region successfully.The S1 gene epitope prediction region Sa(106-1 290 bp)was cloned into the prokaryotic expression vector pET 22b(+)to construct recombi-nant plasmid pET22b-Sa by using the bioinformatics software to analyze the S1 nucleotide and the amino acid sequence.The recombinant expression vector was transformed into Rosetta(DE3)competent strain,and the recombinant protein was acquired by induction of IPTG.The recombi-nant protein was purified by ultrafiltration column concentration and immobilized Ni ion affinity chromatography.The activity of the protein Sa obtained was examined by Western blot.New Zealand rabbits were immunized with the Sa protein 4 times to prepare polyclonal antibody.The results showed that the open reading frame of S1 gene of CHN-2015 strain was 1 566 bp(Gen-Bank No.:KY398009),encoding 522 amino acids.It is a non-transmembrane protein with hydro-philicity and 15 potential B cell epitopes Bit ;The recombinant protein was expressed in prokaryot-ic expression.The protein was mainly expressed in soluble form at 37 ℃ for 5 h at the final con-centration of 0.8 mmol·L －1IPTG.Immunoblotting results showed that the expressed Sa protein had good immunogenicity in the supernatant and inclusion bodies.T he rabbit anti-Sa antibody titer was up to 1 :10 240.The results showed that the expressed Sa protein contained antigen epitope recognition region of porcine Deltacoronavirus,and had a good immunogenicity.