Ⅱ型草鱼呼肠孤病毒VP4、VP35蛋白多克隆抗体制备及其免疫原性分析
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国家水生动物病原库,国家水生动物病原库,国家水生动物病原库

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现代农业产业技术体系建设专项(CARS-46-12)


Preparation and immunogenicity of polyclonal antibodies against VP4, VP35 protein of type Ⅱ grass carp reovirus
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National pathogen collection center for aquatic animals,College of Fisheries and Life Science,Shanghai Ocean University,National pathogen collection center for aquatic animals,College of Fisheries and Life Science,Shanghai Ocean University,National pathogen collection center for aquatic animals,College of Fisheries and Life Science,Shanghai Ocean University

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    摘要:

    为建立针对Ⅱ型草鱼呼肠孤病毒(GCRV)的血清学检测方法,分别构建了GCRVJX02 株外衣壳蛋白VP4、VP35的原核重组表达质粒PGEX-4T-3-S6、PGEX-4T-3-S11,用纯化的重组蛋白rVP4、rVP35分别免疫小鼠制得相应的多克隆抗体,用间接ELISA方法测定2种抗体的效价,用Western Blot鉴定抗体的特异性。SDS-PAGE分析细菌表达的rVP4、rVP35大小分别约为98ku和61ku,且都主要以包涵体的形式存在;间接ELISA方法测定制备的抗体效价分别约为1:4×105和1:106;Western Blot结果显示,制备的2种多克隆抗体都既能够识别原核表达的重组蛋白,又能够识别JX02毒株上的对应蛋白,并且发现感染JX02的草鱼血清中存在结合VP4、VP35的相应抗体。本研究制备的2种多克隆抗体都具有良好的生物学特性,并且这2种重组蛋白作为相应抗体捕获原可以用于通过检测抗病毒抗体来确诊草鱼是否感染Ⅱ型GCRV。本研究将为GCRV主要流行株血清学检测方法的建立以及VP4、VP35蛋白相关功能研究奠定基础。

    Abstract:

    To develop serological methods for detecting type Ⅱ grass carp reovirus (GCRV), the genes encoding outer capsid proteins VP4 and VP35 were cloned into the prokaryotic expression vector PGEX-4T-3 respectively. Polyclonal antibodies were generated by immunization of mices with the purified recombinant VP4 and VP35 proteins respectively and titered by ELISA.The specificity of antibodies was determined by Western blot. SDSPAGE results showed that the size of the recombinant VP4 and VP35 proteins was about 98ku and 61ku, respectively. The recombinant proteins mainly existed in the form of inclusion bodies. The titer of antibody against VP4 was up to 1:4×105, the titer of antibody against VP35 was up to 1:106. The results of Western blot showed that antibodies were able to identify both recombinant proteins and corresponding capsid proteins in virus particles. Furthermore, serum antibodies of grass carp infected by JX02 were able to recognize VP4 and VP35 proteins specifically. In this paper, the polyclonal antibodies against VP4 and VP35 were successfully prepared, which laid a foundation for developing a serological diagnostic method for major epidemic strains of GCRV and functional studies of these two viral capsid proteins.

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宗乾坤,张也,吕利群.Ⅱ型草鱼呼肠孤病毒VP4、VP35蛋白多克隆抗体制备及其免疫原性分析[J].水产学报,2016,40(3):355~362

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  • 收稿日期:2015-11-16
  • 最后修改日期:2016-01-14
  • 录用日期:2016-03-22
  • 在线发布日期: 2016-03-29
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