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CN 41-1437/TS  ISSN 2096-1553

采用狂犬病毒G基因原核表达的间接ELISA方法

李胜利 ,  于向东 ,  王伟杰 ,  胡益源

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李胜利, 于向东, 王伟杰, 等. 采用狂犬病毒G基因原核表达的间接ELISA方法[J]. 轻工学报, 2014, 29(4): 24-28. doi: 10.3969/j.issn.2095-476X.2014.04.006
引用本文: 李胜利, 于向东, 王伟杰, 等. 采用狂犬病毒G基因原核表达的间接ELISA方法[J]. 轻工学报, 2014, 29(4): 24-28. doi: 10.3969/j.issn.2095-476X.2014.04.006
shu
LI Sheng-li, YU Xiang-dong, WANG Wei-jie and et al. Construction of indirect ELISA method using rabies virus G gene expressed in prokaryotic system[J]. Journal of Light Industry, 2014, 29(4): 24-28. doi: 10.3969/j.issn.2095-476X.2014.04.006
Citation: LI Sheng-li, YU Xiang-dong, WANG Wei-jie and et al. Construction of indirect ELISA method using rabies virus G gene expressed in prokaryotic system[J]. Journal of Light Industry, 2014, 29(4): 24-28. doi: 10.3969/j.issn.2095-476X.2014.04.006
shu

采用狂犬病毒G基因原核表达的间接ELISA方法

  • 济源市动物卫生监督所, 河南 济源 459000;

  • 洛阳市动物疫病预防控制中心, 河南 洛阳 471002

  • 中图分类号: R446.6

Construction of indirect ELISA method using rabies virus G gene expressed in prokaryotic system

  • Jiyuan Institute of Animal Health Inspection, Jiyuan 459000, China;

  • Luoyang Center for Animal Disease Control and Prevention, Luoyang 471002, China

  • Received Date: 2014-04-21
    Available Online: 2014-07-15

    CLC number: R446.6

  • 摘要: 为获得狂犬病毒G基因并在大肠杆菌中进行表达,进而初步建立用于评价狂犬疫苗免疫效果的方法,根据狂犬病病毒RV(rabies virus) ERA株G基因序列设计引物,从病毒中提取出RNA,经逆转录并扩增得到RVG基因;将该基因片段定向克隆到原核表达载体pET28a(+)中,构建重组质粒PET-RVG并转化到大肠杆菌BL21(DE3)gold中,经IPTG诱导表达并确定表达的最佳条件;Ni亲和层析柱纯化获得重组G蛋白,并以G蛋白为抗原建立检测狂犬病毒抗体的间接ELISA方法.检测结果表明,经灰度分析纯化后纯度可达96%.
    Abstract: In order to acquire rabies virus G gene and express it in E.coli and establish the evaluation method for rabies virus immune, according to Rabies virus ERA G gene sequence to design primer sequences,RNA was extracted from rabies virus,the first strand cDNA was synthesized by reverse transcription.G gene fragments were amplified by PCR.Then the gene fragment was cloned to the prokaryotic expression vector pET28a (+) to construct PET-RVG.The positive recombinant plasmids were transfected into E.coli BL21(DE3)gold,with IPTG induced expression and determined the best expression condition.Using Niaffinity chromatography purification with G protein,and the indirect ELISA assay for the detection of rabies virus antibodies in serum was established based on the G protein.The results showed that the purity degree was 96%.
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  • 收稿日期:  2014-04-21
  • 刊出日期:  2014-07-15
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李胜利, 于向东, 王伟杰, 等. 采用狂犬病毒G基因原核表达的间接ELISA方法[J]. 轻工学报, 2014, 29(4): 24-28. doi: 10.3969/j.issn.2095-476X.2014.04.006
引用本文: 李胜利, 于向东, 王伟杰, 等. 采用狂犬病毒G基因原核表达的间接ELISA方法[J]. 轻工学报, 2014, 29(4): 24-28. doi: 10.3969/j.issn.2095-476X.2014.04.006
shu
LI Sheng-li, YU Xiang-dong, WANG Wei-jie and et al. Construction of indirect ELISA method using rabies virus G gene expressed in prokaryotic system[J]. Journal of Light Industry, 2014, 29(4): 24-28. doi: 10.3969/j.issn.2095-476X.2014.04.006
Citation: LI Sheng-li, YU Xiang-dong, WANG Wei-jie and et al. Construction of indirect ELISA method using rabies virus G gene expressed in prokaryotic system[J]. Journal of Light Industry, 2014, 29(4): 24-28. doi: 10.3969/j.issn.2095-476X.2014.04.006
shu

采用狂犬病毒G基因原核表达的间接ELISA方法

  • 济源市动物卫生监督所, 河南 济源 459000;
  • 洛阳市动物疫病预防控制中心, 河南 洛阳 471002
  • 收稿日期: 2014-04-21
  • 网络出版日期: 2014-07-15

摘要: 为获得狂犬病毒G基因并在大肠杆菌中进行表达,进而初步建立用于评价狂犬疫苗免疫效果的方法,根据狂犬病病毒RV(rabies virus) ERA株G基因序列设计引物,从病毒中提取出RNA,经逆转录并扩增得到RVG基因;将该基因片段定向克隆到原核表达载体pET28a(+)中,构建重组质粒PET-RVG并转化到大肠杆菌BL21(DE3)gold中,经IPTG诱导表达并确定表达的最佳条件;Ni亲和层析柱纯化获得重组G蛋白,并以G蛋白为抗原建立检测狂犬病毒抗体的间接ELISA方法.检测结果表明,经灰度分析纯化后纯度可达96%.

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