草鱼呼肠孤病毒GCRV-GD108株VP5蛋白功能及免疫原性分析
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中国水产科学研究院珠江水产研究所,农业部热带亚热带水产资源利用与养殖重点实验室,上海海洋大学水产与生命学院;中国水产科学研究院珠江水产研究所,农业部热带亚热带水产资源利用与养殖重点实验室,上海海洋大学水产与生命学院;中国水产科学研究院珠江水产研究所,农业部热带亚热带水产资源利用与养殖重点实验室,中国水产科学研究院珠江水产研究所,农业部热带亚热带水产资源利用与养殖重点实验室,中国水产科学研究院珠江水产研究所,农业部热带亚热带水产资源利用与养殖重点实验室,中国水产科学研究院珠江水产研究所,农业部热带亚热带水产资源利用与养殖重点实验室,中国水产科学研究院珠江水产研究所

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广东省重点科技项目(2008A020100016);广东省海洋渔业科技项目(A200899F01,A201101G01)


Analysis of function and immunogenicity of GCRV-GD108 VP5
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Pearl River Fishery Research Institute,CAFS,College of Fisheries Life,Shanghai Ocean University;Pearl River Fishery Research Institute,CAFS,College of Fisheries Life,Shanghai Ocean University;Pearl River Fishery Research Institute,CAFS,Pearl River Fishery Research Institute,CAFS,Pearl River Fishery Research Institute,CAFS,Pearl River Fishery Research Institute, CAFS; Key Lab of Tropic,Pearl River Fishery Research Institute, CAFS

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2008A020100016;A200899F01、A201101G01;2009J1-C021、20084411115

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    摘要:

    在前期研究中分离到一株草鱼呼肠孤病毒,GCRV-GD108,并获得其全基因组序列。该病毒株的M5基因编码VP5蛋白,该蛋白与哺乳动物正呼肠孤病毒μ2蛋白具有较高的同源性及相似的NTPase结合保守区域,推测VP5蛋白也同样具有NTPase活性。为检测VP5蛋白是否具有NTPase活性及其是否具有免疫保护作用,采用已构建的原核表达载体表达VP5重组蛋白,通过孔雀绿钼酸铵法检测纯化后的重组蛋白的NTPase活性。采用 DNAstar软件预测M5基因编码蛋白的抗原性,综合蛋白亲水性、表面可及性与表面抗原性三项指标,预测编码蛋白可形成抗原表位的氨基酸区域数多达86个,提示VP5蛋白具有较强的免疫原性。用重组蛋白VP5免疫健康草鱼,通过人工攻毒实验检测VP5蛋白的免疫保护作用。结果显示,VP5重组蛋白具有NTPase活性,且其NTPase活性依赖于Mg2+或Na+/K+,而Ca2+的存在可能抑制其活性;VP5蛋白可诱导草鱼产生高水平的抗体滴度,并显著提高IgM mRNA的表达水平,但未能为草鱼提供抗GCRV感染的保护。研究首次证实GCRV-GD108株VP5蛋白具有NTPase活性,但不能为草鱼提供免疫保护作用。

    Abstract:

    A strain of grass carp reovirus,named GCRV-GD108 was isolated from sickened grass carp with symptoms of haemorrhage in Guangdong Province.Its whole genomic sequence was obtained.This reovirus consisted of 11 dsRNA segments which encoded 11 proteins instead of 12 proteins that belonged to Aquareovirus(AQRV).Sequence comparison showed that it possessed only 7 homologous proteins to grass carp reovirus(GCRV)(with 17.6%-45.8% identities),but 9 homologs to MRV(with 15%-46% identities).The virus structure protein VP5,which was encoded by M5 gene of GCRV-GD108,shared a high homology and a conserved motif for NTP binding with the μ2 protein of mammalian reovirus(MRV),suggesting that the VP5 protein is functional homologue of μ2,and possesses NTPase activity.A prokaryotic expression vector of M5 gene had been constructed previously,and the engineering bacteria pET30c-M5/BL21(DE3)was selected.According to the requirements of experience,we had chosen appropriate purification method of VP5 protein.The NTPase activity of the purified recombinant VP5 protein was analyzed,using the method of malachite green/ammonium molybdate reagent for quantification of the released Pi of NTP.The result showed that the recombinant protein possessed NTPase activity.The activity of VP5 is dependent on the cations Mg2+ or Na+/K+,but its activity was inhibited with the presence of Ca2+.DNAstar software was used to predict the antigenicity of M5 gene encoded protein.Online prediction based on the analysis results of the three indexes,hydrophilicity,surface probability and antigenic index,showed that 86 regions in the M5 encoding protein potentially form epitopes,suggesting that VP5 possesses strong immunogenicity.The purified recombinant VP5 protein was used to immunize the healthy grass carp,and its protection role was tested by artificial infection of the immunized fish.The serum of the immunized grass carp was analyzed by ELISA and the mRNA level of IgM from the head kidney of grass carp was analyzed by qRT-PCR.These results indicated that recombinant protein VP5 could induce the immunized grass carp to produce high titer antibodies and the expression levels of IgM were significantly improved.However,this protein could not provide protection for GCRV infection.The NTPase activity of GCRV-GD108 VP5 protein was confirmed for the first time,but this protein could not provide immune protection for grass carp.

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王杭军,叶星,田园园,张莉莉,邓国成.草鱼呼肠孤病毒GCRV-GD108株VP5蛋白功能及免疫原性分析[J].水产学报,2013,37(1):109~116

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  • 收稿日期:2012-05-23
  • 最后修改日期:2012-10-05
  • 录用日期:2012-12-02
  • 在线发布日期: 2013-01-15
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