虹鳟IgM重链恒定区的表达及兔抗血清的制备
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中国水产科学研究院 黑龙江水产研究所,中国水产科学研究院 黑龙江水产研究所,中国水产科学研究院 黑龙江水产研究所,中国水产科学研究院 黑龙江水产研究所,中国水产科学研究院 黑龙江水产研究所,中国水产科学研究院 黑龙江水产研究所,中国水产科学研究院 黑龙江水产研究所

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Q 785; S 917

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国家科技支撑计划(2012BAD25B02);中央级公益性科研院所基本科研业务费专项(HSY201204);黑龙江省冷水性鱼类种质资源及增养殖重点开放实验室(201104)


Expression and rabbit antisera preparation of IgM heavy chain gene in rainbow trout(Oncorhynchus mykiss)
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Heilongjiang River Fishery Research Institute Chinese Academy of Fishery Sciences,Heilongjiang River Fishery Research Institute Chinese Academy of Fishery Sciences,Heilongjiang River Fishery Research Institute Chinese Academy of Fishery Sciences,Heilongjiang River Fishery Research Institute Chinese Academy of Fishery Sciences,Heilongjiang River Fishery Research Institute Chinese Academy of Fishery Sciences,Heilongjiang River Fishery Research Institute Chinese Academy of Fishery Sciences,Heilongjiang River Fishery Research Institute Chinese Academy of Fishery Sciences

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    摘要:

    为了能够成功表达虹鳟IgM,本研究利用生物信息学软件对虹鳟IgM的亲水性及抗原性进行了分析,根据GenBank收录的虹鳟IgM重链恒定区,参照生物信息学分析结果,设计用于扩增截短的IgM基因的引物,以虹鳟头肾RNA提取物为模板,利用RT-PCR方法扩增虹鳟截短的IgM重链恒定区部分基因片段,连接原核表达载体pET-27b,利用大肠杆菌Rosetta进行表达。SDS-PAGE及HPLC结果显示,纯化后截短的IgM大小约为47.7 ku,且纯度达到90%。利用其制备兔抗血清后ELISA分析结果显示,所制备的兔抗血清与本研究所表达的截短IgM蛋白的反应效价为1:40 000,与虹鳟血清提取的全长IgM反应效价为1:20 000并且呈现出抗原计量依赖性。研究表明,重组IgM蛋白与虹鳟血清中的天然IgM重链恒定区具有近似的结构,利用其所制备的兔抗血清能够与虹鳟鱼体中的天然IgM发生特异性反应。

    Abstract:

    Rainbow trout, Oncorhynchus mykiss, is one of the cold water fishes cultured in our country, and the intensification of culture of rainbow trout led to outbreak of several diseases which made the immune system study a new research field.In this study, the truncated heavy chain of IgM was obtained by RT-PCR from rainbow trout head-kidney RNA extracts and cloned into pET-27b vector.The predicted molecular weight of recombinant IgM was approximately 47.7 ku.After purification, the purity of recombinant IgM reached approximately 90% confirmed by SDS-PAGE and HPLC analyses.The rabbit antisera against recombinant truncated IgM in this study could react specifically with both natural IgM from rainbow trout serum and the purified IgM in ELISA test.The titer of rabbit antisera against the recombinant truncated IgM was 1:40 000, the natural IgM was 1:20 000 in an antiserum dose-dependent manner.These results indicated that the recombinant IgM expressed in this study had the comparable structure with the natural IgM of the rainbow trout serum.The results provide important experimental basis for trout immune system study and related immunodetection of rainbow trout.

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赵景壮,徐黎明,刘淼,曹永生,尹家胜,刘红柏,卢彤岩.虹鳟IgM重链恒定区的表达及兔抗血清的制备[J].水产学报,2014,38(8):1175~1181

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  • 收稿日期:2014-04-03
  • 最后修改日期:2014-05-15
  • 录用日期:2014-06-20
  • 在线发布日期: 2014-08-21
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