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山羊痘病毒糖蛋白基因ORF112的原核表达及多克隆抗体的制备
引用本文:郑敏,李春艳,陆文俊,莫胜兰,屈素洁,粟艳琼,梁媛,施开创,李军.山羊痘病毒糖蛋白基因ORF112的原核表达及多克隆抗体的制备[J].动物医学进展,2012,33(4):1-5.
作者姓名:郑敏  李春艳  陆文俊  莫胜兰  屈素洁  粟艳琼  梁媛  施开创  李军
作者单位:1. 广西动物疫病预防控制中心,广西南宁,530001
2. 新乡市动物卫生监督所,河南新乡,453003
摘    要:为探索山羊痘病毒(GTPV)糖蛋白基因ORF112在疫苗和诊断中的应用,应用PCR技术扩增GTPV弱毒疫苗株AV 41ORF112基因,将其克隆到pET-32a载体,转化感受态细胞BL21,经IPTG诱导后获得与预期大小相符的约37ku的融合蛋白,为可溶性和包涵体表达。应用镍离子亲和树脂对可溶性表达的目的蛋白进行纯化,然后用纯化的融合蛋白免疫Balb/c小鼠,制备多克隆抗体。免疫荧光试验表明该多克隆抗体可以与GTPV反应,为GTPV新型疫苗和诊断试剂的研究奠定了基础。

关 键 词:山羊痘病毒  ORF112蛋白  原核表达  多克隆抗体

Prokaryotic Expression of ORF112 Gene Encoding Glycoprotein of Goatpox Virus and Preparation of Polyclonal Antibodies
ZHENG Min , LI Chun-yan , LU Wen-jun , MO Sheng-lan , QU Su-jie , SU Yan-qiong , LIANG Yuan , SHI Kai-chuang , LI Jun.Prokaryotic Expression of ORF112 Gene Encoding Glycoprotein of Goatpox Virus and Preparation of Polyclonal Antibodies[J].Progress In Veterinary Medicine,2012,33(4):1-5.
Authors:ZHENG Min  LI Chun-yan  LU Wen-jun  MO Sheng-lan  QU Su-jie  SU Yan-qiong  LIANG Yuan  SHI Kai-chuang  LI Jun
Institution:1(1.Guangxi Center for Animal Disease Control and Prevention,Nanning,Guangxi,530001,China; 2.Xinxiang Animal Health Inspection Institute,Xinxiang,Henan,453003,China)
Abstract:To study the potential of the glycoprotein-encoding ORF112 gene of goatpox virus(GTPV) in developments on novel prevention and diagnosis approaches,ORF112 gene was cloned by PCR from genomic DNA of GTPV live vaccine strain AV41,and was subcloned into prokaryotic vector pET-32a to construct recombinant expression plasmid pET-ORF112.The plasmid pET-ORF112 was transformed into competent cells of Escherichia coli BL21 and induced by IPTG.SDS-PAGE analysis showed that the fusion protein was expressed both in soluble type and inclusion body with a molecular weight of 37 ku.Westernblot confirmed that the fusion protein could be recognized by GTPV specific antiserum.Furthermore,the soluble fusion protein was purified by Co ion affinity chromatography purification.Balb/c mice were injected with purified protein and bleed for preparation of ORF112 specific polyclonal antibodies.In the end,specificity and reactogenicity of antibodies were demonstrated by indirect immunofluorescence test.
Keywords:Goatpox virus  ORF112  prokaryotic expression  polyclonal antibody
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