河南农业科学 ›› 2020, Vol. 49 ›› Issue (2): 136-141.DOI: 10.15933/j.cnki.1004-3268.2020.02.018

• 畜牧·兽医 • 上一篇    下一篇

猪瘟病毒Erns蛋白在杆状病毒表达系统中的分泌表达及活性检测

魏蔷1,白怡霖1,2,柴书军1,刘运超1,宋亚鹏1,张改平1,3,4   

  1. (1.河南省农业科学院 动物免疫学重点实验室/河南省动物免疫学重点实验室/农业部动物免疫学重点实验室,河南 郑州 450002;2.西北农林科技大学 动物医学院,陕西 杨陵 712100;3.河南农业大学 牧医工程学院,河南 郑州 450002;4.江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏 扬州 225009)
  • 收稿日期:2019-09-03 出版日期:2020-02-15 发布日期:2020-02-15
  • 通讯作者: 张改平(1960-),男,河南内黄人,研究员,中国工程院院士,博士,主要从事动物免疫学研究。E-mail:zhanggaip@126.com
  • 作者简介:魏蔷(1989-),女,河南驻马店人,助理研究员,博士,主要从事动物免疫学研究。E-mail:chrysqiang@126.com
  • 基金资助:
    国家重点研发计划项目(2017YFD0501103)

Secretory Expression of CSFV Erns Protein in Baculovirus Expression System and Its Activity Analysis

WEI Qiang1,BAI Yilin1,2,CHAI Shujun1,LIU Yunchao1,SONG Yapeng1,ZHANG Gaiping1,3,4   

  1. (1.Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences/Henan Key Laboratory of Animal Immunology/Key Laboratory of Animal Immunology,Ministry of Agriculture,Zhengzhou 450002,China;2.College of Veterinary
    Medicine,Northwest A&F University,Yangling 712100,China; 3.College of Animal Science and Veterinary Medicine,Henan
    Agricultural University,Zhengzhou 450002,China;4.Jiangsu Co-innovation Center for Prevention and Control of Important
    Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)
  • Received:2019-09-03 Published:2020-02-15 Online:2020-02-15

摘要: 为制备用于ELISA检测猪瘟病毒的Erns蛋白,根据昆虫细胞密码子偏好性对Erns的基因序列进行优化,将优化后的Erns基因插入带有信号肽的表达载体pFastBacl-gp67中;随后将重组质粒pFastBacl-gp67-Erns转化至DH10Bac感受态细胞,经蓝白斑筛选,挑选白斑,提取重组黏粒Bacmid-Erns;将重组黏粒转染sf21细胞得到杆状病毒,经扩增后得到P3病毒,利用其大量感染细胞,3 d后收取细胞培养上清;通过镍填料纯化得到分泌表达的Erns蛋白,通过SDS-PAGE及Western blot检测分泌蛋白的表达水平、纯化情况,用PNGase F处理分析其糖基化水平,通过ELISA分析其抗原性。结果显示,成功构建了重组质粒pFastBacl-gp67-Erns,蓝白斑筛选获得了重组黏粒Bacmid-Erns,在此基础上获得了重组杆状病毒,利用其感染sf21细胞,成功分泌表达了Erns蛋白,纯化所得Erns蛋白纯度较高,具有较高糖基化修饰水平,且纯化所得Erns蛋白能特异性识别猪瘟阳性血清,其抗原性良好。

关键词: 猪瘟病毒;Erns蛋白;分泌表达;糖基化分析, 活性, 杆状病毒

Abstract: In order to obtain the CSFV Erns protein for the application in ELISA,the gene sequence of the Erns protein was optimized according to the codon bias of Spodoptera frugiperda cells.Firstly,the gene was inserted into the recombinant vector pFastBacl-gp67,which contains the gp67 signal peptide sequence.Secondly,the resulting plasmid pFastBacl-gp67-Erns was transformed into the DH10Bac competent cells to get the recombinant bacmid through blue-white screening.Thirdly,the recombinant Bacmid-Erns was transfected into the sf21 cells to generate the recombinant baculovirus.Finally,the sf21 cells were infected with the recombinant baculovirus.At three days post infection, culture supernatant was collected.The Erns protein was purified by nickel-affinity chromatography.The expression level and purity of the protein were identified using SDS-PAGE and Western blot analysis.The glycosylation pattern of the purified Erns protein was analyzed using PNGase F.And the indirect ELISA was carried out to analyze the antigenicity of the protein.Consequently,the recombinant pFastBacl-gp67-Erns plasmid was generated.And the Bacmid-Erns was obtained using blue-white screening.The recombinant baculovirus was used for large-scale expression of Erns.The secretory expression of Erns was achieved using signal peptide.It was shown that the purified Erns was of high purity.And the Erns was glycosylated properly.The purified Erns protein could be specifically recognized by the CSFV positive serum,indicating that the purified protein was of high activity.

Key words: Classical swine fever virus(CSFV), Erns protein;Secretory expression;Glycosylation analysis;Activity ;Baculovirus

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