畜牧兽医学报2018，Vol.49Issue(6)：1256-1264,9.DOI:10.11843/j.issn.0366-6964.2018.06.018
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
Cloning and Prokaryotic Expression of Porcine Deltacoronavirus S1 Gene and Preparation of Polyclonal Antibody
摘要
Abstract
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.关键词
猪丁型冠状病毒/S1基因/克隆/原核表达/抗原性/多克隆抗体Key words
porcine deltacoronavirus/S1 gene/clone/prokaryotic expression/antigenicity/poly-clonal antibody分类
农业科技引用本文复制引用
李成,赵勤,伍锐,张雨迪,黄小波,刘浩宇,刘志鹏,赵玉佳,曹三杰,文心田,文翼平..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[J].畜牧兽医学报,2018,49(6):1256-1264,9.基金项目
国家重点研发计划(2016YFD0500700) （2016YFD0500700）
