| 新城疫病毒ClassⅠ强毒株L基因的表达及单克隆抗体的制备 |
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| 引用本文： | 韦娜娜,于洋,陈鸿军,仇旭升,于圣青,宋翠萍,谭磊,任涛,丁铲.新城疫病毒ClassⅠ强毒株L基因的表达及单克隆抗体的制备[J].中国兽医科学,2012(4):347-351. |
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| 作者姓名： | 韦娜娜 于洋 陈鸿军 仇旭升 于圣青 宋翠萍 谭磊 任涛 丁铲 |
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| 作者单位： | 华南农业大学兽医学院农业部动物疫病防控重点开放实验室广东省动物源性人畜共患病预防与控制重点实验室;中国农业科学院上海兽医研究所;国家家禽工程技术研究中心 |
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| 基金项目： | 国家自然科学基金青年基金项目(31101822);公益性行业(农业)专项(201003012) |
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| 摘 要： | 采用RT-PCR方法从新城疫病毒(NDV)ClassⅠ强毒株9a5b中扩增出了L基因C末端的基因片段,将其克隆到表达载体pGEX-6P-1中,测序验证后转化入表达宿主菌BL21进行IPTG诱导表达,表达的蛋白接种8周龄BALB/c小鼠,制备L蛋白的单克隆抗体,用间接免疫荧光试验(IFA)、酶联免疫吸附试验(ELISA)和Western-bolt方法共同检测所获得的抗体。结果显示,重组菌可表达出分子质量约为39ku的重组融合蛋白。SDS-PAGE分析显示,表达的蛋白以包涵体的形式存在于菌体中。多种方法筛选显示成功制备了4株单克隆抗体。免疫特异性鉴定结果表明,在所获得的单抗中,4株单抗均具有ELISA和IFA效价,且4株单抗与ClassⅠ毒株的反应均强于与ClassⅡ毒株的反应。
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| 关 键 词： | 新城疫病毒 原核表达 单克隆抗体 L蛋白 杂交瘤细胞 |
Preparation of the monoclonal antibodies of against L protein of ClassⅠ Newcastle disease virus virulent strain |
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| WEI Na-na,YU Yang,CHEN Hong-jun,QIU Xu-sheng,YU Sheng-qing,SONG Cui-ping,TAN Lei,REN Tao,DING Chan.Preparation of the monoclonal antibodies of against L protein of ClassⅠ Newcastle disease virus virulent strain[J].Veterinary Science in China,2012(4):347-351. |
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| Authors: | WEI Na-na YU Yang CHEN Hong-jun QIU Xu-sheng YU Sheng-qing SONG Cui-ping TAN Lei REN Tao DING Chan |
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| Institution: | 2,3(1.Key Laboratory of Animal Disease Control and Prevention of the Ministry of Agriculture/Key Laboratory of Zoonoses Control and Prevention of Guangdong/College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;2.Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences, Shanghai 200241,China;3.National Poultry Technology Research Center,Shanghai 200331,China) |
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| Abstract: | L gene of ClassⅠ9a5b virulent strain was amplified by RT-PCR and cloned to pGEX-6P-1 expression vector.After sequencing,the recombinant plasmid was transformed into BL21 which were then induced by IPTG to produce a recombinant protein of 39 ku.BALB/c mice were immunized with the purified expression product of L protein.The ELISA,indirect immunofluorescence assay IFA and Western-blot analysis were used to characterize and identify antibodies.Results showed that a panel of 4 hybridoma cell lines were successfully prepared.All the 4 McAbs had the ability of ELISA and IFA,and all the 4 McAbs had stronger reaction with ClassⅠ than ClassⅡ. |
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| Keywords: | Newcastle disease virus prokaryotic expression monoclonal antibody L protein hybridoma cell |
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