畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (6): 1256-1264.doi: 10.11843/j.issn.0366-6964.2018.06.018

• 预防兽医 • 上一篇    下一篇

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

李成, 张雨迪, 黄小波*, 刘浩宇, 刘志鹏, 赵玉佳, 曹三杰, 文心田, 文翼平, 赵勤, 伍锐   

  1. 四川农业大学动物医学院猪病研究中心, 成都 611130
  • 收稿日期:2017-10-19 出版日期:2018-06-23 发布日期:2018-06-23
  • 通讯作者: 黄小波,教授,博士生导师,E-mail:rsghb110@126.com
  • 作者简介:李成(1991-),男,四川南充人,硕士,主要从事动物传染病的研究工作,E-mail:azjajr0918@qq.com
  • 基金资助:

    国家重点研发计划(2016YFD0500700)

Cloning and Prokaryotic Expression of Porcine Deltacoronavirus S1 Gene and Preparation of Polyclonal Antibody

LI Cheng, ZHANG Yu-di, HUANG Xiao-bo*, LIU Hao-yu, LIU Zhi-peng, ZHAO Yu-jia, CAO San-jie, WEN Xin-tian, WEN Yi-ping, ZHAO Qin, WU Rui   

  1. Research Center of Swine Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
  • Received:2017-10-19 Online:2018-06-23 Published:2018-06-23

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。

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 pET22b(+) to construct recombinant 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 recombinant 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 (GenBank No.:KY398009), encoding 522 amino acids. It is a non-transmembrane protein with hydrophilicity and 15 potential B cell epitopes Bit;The recombinant protein was expressed in prokaryotic expression. The protein was mainly expressed in soluble form at 37℃ for 5 h at the final concentration of 0.8 mmol·L-1 IPTG. Immunoblotting results showed that the expressed Sa protein had good immunogenicity in the supernatant and inclusion bodies. The 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.

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