鹅细小病毒NS1蛋白的表达及ELISA方法的建立
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- 英文论文题名：Expression and Establishment of an ELISA Assay for the NS1 Protein of Goose Parvovirus
- 论文作者：孙敏华 ; 董嘉文 ; 李林林 ; 袁建丰 ; 邝瑞欢 ; 张春红 ; 郭鹏举 ; 胡奇林 ; 张建峰
- 英文论文作者：Sun Minhua ; Dong Jiawen ; Li Linlin ; Yuan Jianfeng ; Kuang Ruihuan ; Zhang Chunhong ; Guo Pengju ; Hu Qilin ; Zhang Jianfeng ; Guangdong Open Laboratory of Public Health ; Institute of Animal Health ; Guangdong Academy of Agricultural Sciences
- 年：2016
- 作者机构：广东省农业科学院动物卫生研究所广东省兽医公共卫生公共实验室;
- 论文关键词：鹅细小病毒 ; NS1蛋白 ; ELISA
- 英文论文关键词：Goose Parvovirus ; NS1 protein ; ELISA
- 会议召开时间：2016-06-14
- 会议录名称：第25届广东省科技进步活动月畜牧兽医学术与科技创新发展大会论文集
- 语种：中文
- 分类号：S852.65
- 学会代码：GDKL
- 会议名称：第25届广东省科技进步活动月畜牧兽医学术与科技创新发展大会
- 会议地点：中国广东广州
- 主办单位：广东省畜牧兽医学会
- 学会名称：广东省科学技术协会科技交流部
- 页数：6
- 文件大小：577k
- 原文格式：O
- 会议级别：地方
摘要
为了监测番鸭群中鹅细小病毒(Goose Parvovirus,GPV)的血清学状况,本研究表达了鹅细小病毒NS1蛋白,并建立了检测番鸭血清中鹅细小病毒抗体的间接ELISA方法。通过PCR方法克隆了NS1基因,经亚克隆至pET32a表达载体,该重组质粒经IPTG诱导表达蛋白的相对分子质量约为92 kDa。Wes tern blot分析表明,该重组蛋白能与鹅细小病毒弱毒疫苗免疫的番鸭阳性血清发生特异性反应。随后,通过棋盘法确定了当纯化的NS1蛋白包被量为0.5μg/孔,待检血清1:50稀释,HRP标记的兔抗鸭二抗1:1 0000倍稀释时反应条件最佳。在最佳反应条件下,阴阳性临界值判定标准为0.399。该方法特异性强,与番鸭细小病毒、鸭瘟病毒、鸭肝炎病毒、新城疫病毒和鸭疫里默氏杆菌的阳性血清均无交叉反应;重复性高,批内和批间重复试验的变异系数分别小于5%和10%。用建立的ELISA方法检测养殖场中免疫过鹅细小病毒的番鸭血清样品88份,测得81份为阳性,阳性率为92%。该方法为鹅细小病毒的血清学诊断和流行病学监测奠定了基础。
To survillance the serological prevalence of Goose parvovirus antibodies in muscovy duck flocks,an indirect ELISA(Enzyme Linked Immunosorbent Assay) based on the recombinant NS1 protein was established.The NS1 gene was amplified by PCR and subcloned into pET32 a vector,then a protein about 92 kilodalton was expressed by IPTG induction.Western blot analysis showed the recombinant NS1 protein reacted well with the positive serum which collected from the inmmuned muscovy duck.Checkboard assay showed when NS1 protein coated with a dosage of 0.5 microgram per well(μg/weH),the positive serum undergone 1:50 dilution,and HRP labelled antibody diluted to 1:10000,the assay was optimal with a cut-off value of 0.399.The high specificity made the assay reacted with none of the these positive sera,including Muscovy duck parvovirus,Duck plague virus,Duck hepatitis virus,Newcastle disease virus and Riemerella anatipestifer.The intra- and inter- coefficient of variabilities were 5%and 10%respectively,which suggested the assay was stable.Clinical tests showed that 81 of 88 sera collected from immuned muscovy ducks were positive,thus the positive rate was 92%.In conclusion,this method could be useful for the serological detection and survillence of Goose parvovirus.
To survillance the serological prevalence of Goose parvovirus antibodies in muscovy duck flocks,an indirect ELISA(Enzyme Linked Immunosorbent Assay) based on the recombinant NS1 protein was established.The NS1 gene was amplified by PCR and subcloned into pET32 a vector,then a protein about 92 kilodalton was expressed by IPTG induction.Western blot analysis showed the recombinant NS1 protein reacted well with the positive serum which collected from the inmmuned muscovy duck.Checkboard assay showed when NS1 protein coated with a dosage of 0.5 microgram per well(μg/weH),the positive serum undergone 1:50 dilution,and HRP labelled antibody diluted to 1:10000,the assay was optimal with a cut-off value of 0.399.The high specificity made the assay reacted with none of the these positive sera,including Muscovy duck parvovirus,Duck plague virus,Duck hepatitis virus,Newcastle disease virus and Riemerella anatipestifer.The intra- and inter- coefficient of variabilities were 5%and 10%respectively,which suggested the assay was stable.Clinical tests showed that 81 of 88 sera collected from immuned muscovy ducks were positive,thus the positive rate was 92%.In conclusion,this method could be useful for the serological detection and survillence of Goose parvovirus.
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