| 乙型脑炎病毒E蛋白基因的原核表达及ELISA抗体检测方法的建立 |
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| 引用本文： | 刘昊,尹革芬,鲁会军,张丹,谭磊,王开艳,关莎莎,段博芳,金宁一,段纲.乙型脑炎病毒E蛋白基因的原核表达及ELISA抗体检测方法的建立[J].中国兽医科学,2010,40(4). |
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| 作者姓名： | 刘昊 尹革芬 鲁会军 张丹 谭磊 王开艳 关莎莎 段博芳 金宁一 段纲 |
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| 作者单位： | 刘昊,LIU Hao(云南农业大学动物科技学院,云南昆明,650201;中国人民解放军军事医学科学院军事兽医研究所,吉林长春,130062);尹革芬,关莎莎,段博芳,段纲,YIN Ge-fen,GUAN Sha-sha,DUAN Bo-fang,DUAN Gang(云南农业大学动物科技学院,云南昆明,650201);鲁会军,金宁一,LU Hui-jun,JIN Ning-yi(中国人民解放军军事医学科学院军事兽医研究所,吉林长春,130062);张丹,谭磊,ZHANG Dan,TAN Lei(中国人民解放军军事医学科学院军事兽医研究所,吉林长春,130062;吉林大学畜牧兽医学院,吉林长春,130062);王开艳,WANG Kai-yan(延边大学农学院动物医学系,吉林龙井,133400) |
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| 基金项目： | 国家公益性行业科研专项，国家高技术研究发展计划(863计划) |
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| 摘 要： | 通过PCR方法扩增流行性乙型脑炎病毒(JEV)E基因,全长1 500 bp,将其连入经双酶切的pET-28a(+)载体,构建了重组原核表达质粒pET28a-E.将pET28a-E转化大肠杆菌BL21(DE3)后,经IPTG诱导,进行SDS-PAGE分析.结果显示,E基因在大肠杆菌BL21中获得高效表达,表达的蛋白分子质量约53 ku.Western-blot分析表明,该表达产物具有良好的抗原性.在此基础上,利用该蛋白初步建立了检测猪JEV抗体的间接ELISA方法,并用武汉科前生物制品公司生产的猪JEV ELISA抗体检测试剂盒同时对250份临床采集的猪血清样品进行了检测,结果这两种方法的符合率达到92%,表明建立的ELISA方法具有较高的灵敏性和特异性.
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| 关 键 词： | 乙型脑炎病毒 E基因 克隆 表达 抗原性 |
Prokaryotic expression of E protein gene of Japanese encephalitis virus (JEV) and establishment of an indirect ELISA for detection of antibody against JEV |
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| LIU Hao,YIN Ge-fen,LU Hui-jun,ZHANG Dan,TAN Lei,WANG Kai-yan,GUAN Sha-sha,DUAN Bo-fang,JIN Ning-yi,DUAN Gang.Prokaryotic expression of E protein gene of Japanese encephalitis virus (JEV) and establishment of an indirect ELISA for detection of antibody against JEV[J].Veterinary Science in China,2010,40(4). |
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| Authors: | LIU Hao YIN Ge-fen LU Hui-jun ZHANG Dan TAN Lei WANG Kai-yan GUAN Sha-sha DUAN Bo-fang JIN Ning-yi DUAN Gang |
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| Institution: | LIU Hao1,2,YIN Ge-fen1,LU Hui-jun2,ZHANG Dan2,3,TAN Lei2,WANG Kai-yan4,GUAN Sha-sha1,DUAN Bo-fang1,JIN Ning-yi2,DUAN Gang1(1.College of Animal Science , Technology,Yunnan Agricultural University,Kunming 650201,China,2.Institute of Military Veterinary,PLA Academy of Military Medical Sciences,Changchun 130062,3.College of Animal Science , Veterinary Medicine,Jilin University,4.Department of Veterinary Medicine,College of Agriculture,Yanbian University,Longjing 133400,China) |
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| Abstract: | According to the E gene sequence of Japanese encephalitis virus(JEV),a pair of primers was designed.With the primers,the objective gene was amplified with 1500bp in size.Then the amplified E gene was cloned into prokaryotic expression vector pET-28a(+) to construct recombinant plasmid.The plasmid was transformed into BL21(DE3) competent cell,and the E protein was expressed highly with 53ku approximately in molecular weight.Western-blot analysis showed that E protein could react with positive swine sera.Base... |
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| Keywords: | Japanese encephalitis virus(JEV) E gene cloning expression antigenicity |
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