畜牧兽医学报2024,Vol.55Issue(10) :4773-4778.DOI:10.11843/j.issn.0366-6964.2024.10.046

猫传染性腹膜炎病毒N蛋白表达及其多克隆抗体制备

Preparation and Application of N Protein Polyclonal Antibody of Feline Infectious Peritonitis Virus SH2021 Strain

刘福康 袁莉刚 张达 汤傲星 刘光清 朱杰
畜牧兽医学报2024,Vol.55Issue(10) :4773-4778.DOI:10.11843/j.issn.0366-6964.2024.10.046

猫传染性腹膜炎病毒N蛋白表达及其多克隆抗体制备

Preparation and Application of N Protein Polyclonal Antibody of Feline Infectious Peritonitis Virus SH2021 Strain

刘福康 1袁莉刚 2张达 3汤傲星 3刘光清 3朱杰3
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作者信息

  • 1. 甘肃农业大学动物医学院,兰州 730070;中国农业科学院上海兽医研究所,上海 200241
  • 2. 甘肃农业大学动物医学院,兰州 730070
  • 3. 中国农业科学院上海兽医研究所,上海 200241
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摘要

本研究旨在体外原核表达猫传染性腹膜炎病毒(feline infectious peritonitis virus,FIPV)SH2021株核衣壳蛋白(nucleocapsid,N),制备兔源多克隆抗体.根据FIPV SH2021株N基因序列设计引物,构建pCold-I-N重组表达质粒.随后,将重组质粒转化至表达感受态细胞BL21(DE3),在终浓度为1.0 mmol·L-1的IPTG和16 ℃的诱导条件下进行表达.最后,利用His层析柱进行纯化,纯化后的重组蛋白免疫新西兰大白兔制备多克隆抗体.结果表明,纯化获得重组N蛋白大小约为45 ku,制备的N蛋白兔多克隆抗体效价可达1:512 000,该抗体对N蛋白具有良好的反应原性.本研究成功所制备了 FIPVN蛋白兔多克隆抗体,该抗体具有良好的反应原性和特异性,为FIPV抗原抗体检测以及研究N蛋白生物学功能研究提供重要工具.

Abstract

This study aimed to express the nucleocapsid protein(N)of feline infectious peritonitis virus(FIPV)strain SH2021 in vitro and prepare rabbit polyclonal antibodies.Primers were de-signed based on the N gene sequence of FIPV strain SH2021 to construct the recombinant expres-sion plasmid pCold-I-N.Subsequently,the recombinant plasmid was transformed into expres-sion-competent cells BL21(DE3),and expression was induced under conditions of 1.0 mmol·L-1 IPTG and 16 ℃.Finally,purification was performed using a His column,and the purified re-combinant protein was used to immunize New Zealand white rabbits to prepare polyclonal anti-bodies.The experimental results showed that the purified recombinant N protein had a size of ap-proximately 45 ku,and the titer of the prepared rabbit polyclonal antibodies against the N protein reached 1:512 000.The antibodies exhibited good reactivity and specificity towards the N pro-tein.This study successfully prepared rabbit polyclonal antibodies against the FIPV N protein,which showed good reactivity and specificity,providing important tools for FIPV antigen-anti-body detection and research on the biological functions of the N protein.

关键词

猫传染性腹膜炎病毒/猫冠状病毒/N蛋白/基因克隆/原核表达/多克隆抗体

Key words

feline infectious peritonitis virus/feline coronavirus/N protein/gene cloning/pro-karyotic expression/polyclonal antibody

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基金项目

上海市市级科技重大专项(ZD2021CY001)

上海市自然科学基金项目(22ZR1476300)

出版年

2024
畜牧兽医学报
中国畜牧兽医学会

畜牧兽医学报

CSTPCDCSCD北大核心
影响因子:0.729
ISSN:0366-6964
参考文献量14
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