1型鸭甲肝病毒VP3蛋白的抗血清中和活性分析及B细胞表位鉴定

发布时间：2018-07-03 12:45

本文选题：型鸭甲肝病毒 + VP蛋白　；参考：《畜牧兽医学报》2016年01期

【摘要】：旨在探究1型鸭甲肝病毒(DHAV-1)VP3蛋白抗血清的中和活性并鉴定VP3的线性B细胞表位。利用pGEX-4T-1表达载体,在BL21(DE3)宿主菌中原核表达DHAV-1 VP3基因,以切胶纯化出的蛋白质为抗原免疫兔制备多克隆抗体,通过鸡胚中和试验对多抗的中和效价进行检测;采用KarplusSchulz、Emini、Jameson-Wolf和Parker方法分别对柔韧性、表面可及性、抗原性及亲水性进行分析,得到了4条候选线性B细胞表位,以制备的兔抗VP3多克隆抗体为一抗,通过间接ELISA方法对人工合成的B细胞表位进行鉴定,并进一步用临床鸭血清样品对鉴定的B细胞表位的抗体检测能力进行评估。结果显示,VP3在BL21(DE3)中以包涵体形式表达,大小约54ku,Western blot分析表明重组蛋白质具有较好的反应原性。制备的兔抗VP3多克隆抗体的琼扩效价达到1∶16,并能中和DHAV-1,中和效价为1∶39;利用间接ELISA鉴定出GKRKPCRRPIHKPKNPPQEP(1—20aa)、FNTGRYQMSWYPIADGEQSL(131—150aa)和VNSSAPSNID(200—209aa)为VP3的B细胞表位,抗体检测能力试验结果显示表位肽可检测临床DHAV-1鸭血清。本研究表明DHAV-1VP3的抗血清具备一定的中和活性,1—20aa、131—150aa和200—209aa为VP3的B细胞表位且具有临床应用前景。
[Abstract]:The aim of this study was to investigate the neutralization activity of DHAV-1 VP3 antiserum and to identify the linear B cell epitopes of VP3. Using pGEX-4T-1 expression vector, DHAV-1 VP3 gene was expressed in BL21 (DE3) host strain. The polyclonal antibody was prepared by immunizing rabbits with the purified protein. The neutralization titer of the polyclonal antibody was detected by chicken embryo neutralization test. The flexibility, surface accessibility, antigenicity and hydrophilicity of four candidate linear B cell epitopes were analyzed by Karplus Schulzian minieson-Wolf and Parker methods respectively. The prepared rabbit anti-VP3 polyclonal antibody was used as the first antibody. The synthetic B cell epitopes were identified by indirect Elisa, and the antibody detection ability of the identified B cell epitopes was further evaluated with clinical duck serum samples. The results showed that VP3 was expressed as inclusion body in BL21 (DE3). Western blot analysis showed that the recombinant protein had good reactivity. The agarose expansion titer of rabbit anti-VP3 polyclonal antibody was 1: 16, and the neutralization titer of DHAV-1 was 1: 39. GKRKPCRPIHKPKNPPQEP (1-20aa) FNTGRYQMSWYPIADGEQSL (131-150aa) and VNSSAPSNID (200-209aa) were identified as the epitopes of VP3. The results of antibody test showed that epitope peptide could detect clinical DHAV-1 duck serum. The results showed that the antiserum of DHAV-1 VP3 had certain neutralization activity and the B cell epitopes of DHAV-1VP3 and 200-209aa were B cell epitopes of VP3 and had the prospect of clinical application.
【作者单位】：四川农业大学动物医学院禽病防治研究中心;四川农业大学预防兽医研究所;动物疫病与人类健康四川省重点实验室;
【基金】：国家“十二五”科技支撑计划(2015BAD12B05) 国家现代农业(水禽)产业技术体系专项(CARS-43-8) 四川省创新团队项目(12TD005/2013TD0015)
【分类号】：S858.32

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