1型鸭甲肝病毒VP3蛋白间接ELISA方法的建立及其B细胞表位鉴定

发布时间：2018-03-05 13:06

  本文选题：1型鸭甲肝病毒　切入点：VP3蛋白　出处：《四川农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：鸭甲型肝炎病毒(duck hepatitis A virus, DHAV)是一种无囊膜的单股正链RNA病毒,基因组RNA编码结构蛋白和非结构蛋白。其中,结构蛋白对病毒的组装和致病性有重要作用。VP3蛋白是主要的结构蛋白,位于衣壳表面,相对保守,是病毒粒子抗原性的主要决定因子,存在着众多抗原表位,可诱导机体产生免疫应答。1型鸭甲肝病毒(DHAV-1)是分布最广、危害最大的DHAV,本研究对DHAV-1的VP3进行原核表达和纯化,并制备兔抗VP3多克隆抗体,通过鸡胚中和试验探究VP3多抗血清的中和活性；通过间接ELISA对VP3上可能存在的B抗原表位进行鉴定；建立基于VP3的检测DHAV抗体的间接ELISA方法。1. DHAV-1 VP3的原核表达、纯化及鉴定通过RT-PCR得到了预期大小的VP3片段,将目的片段先后T-A克隆至pMD19-T (simple)和亚克隆至pGEX-4T-1,分别构建了重组T质粒pMD19-VP3和重组表达质粒pGEX-VP3。 pGEX-VP3转化BL21(DE3)后表达的重组蛋白约50kD。以0.2mmol/LIPTG、37℃诱导8h表达量最大,且始终以包涵体形式表达。采用切胶纯化获得的重组蛋白纯度高。Western blot表明重组蛋白可被兔抗DHAV-1抗体识别,具有良好的反应原性。2. DHAV-1 VP3的抗血清中和活性分析及B细胞表位鉴定利用纯化的VP3重组蛋白乳剂免疫雄兔,经五免后琼脂扩散试验的效价均达到1：16。Western blot证明兔抗血清可与VP3蛋白特异性结合。鸡胚中和试验表明VP3抗血清的中和效价为2-5.29。运用生物信息学软件综合分析VP3序列的柔韧性、表面可及性、亲水性和抗原性,预测出四条B细胞表位多肽送公司合成。以制备的VP3多克隆抗体作一抗,VP3蛋白作抗原进行间接ELISA,确定了表位肽的最佳稀释度为1：1280,然后以该浓度稀释的表位肽作抗原进行间接ELISA鉴定B细胞表位,结果证明1GKRKPCRRPIHKPKNPPQEP20、 131FNTGRYQMSWYPIADGEQSL150和200VNSSAPSNID209能与VP3多克隆抗体特异性结合,为VP3的B细胞表位。进一步用DHAV-1临床鸭血清样品对B表位的抗体检测能力进行评估,结果表明表位肽可用于检测DHAV-1抗体。3.基于DHAV-1 VP3蛋白的间接ELISA方法的建立通过条件的摸索,得到以VP3作为包被抗原的间接ELISA方法的最佳检测条件为：以9.375 ng/μL蛋白浓度4℃包被过夜,1%明胶37±封闭90min,加1：160稀释的被检血清于37±孵育90min,1：2000稀释的HRP标记兔抗鸭IgG于37±孵育45min。确定了阳性阈值为0.332。该方法有良好的敏感性、重复性及特异性,并能同时检测出DHAV-1和DHAV-3的抗体,与DHAV-1全病毒作抗原的间接ELISA方法的符合率为96%。
[Abstract]:Duck hepatitis A virus (DHAV) is a single-stranded positive strand RNA virus with no envelope. The genomic RNA encodes structural and non-structural proteins. Structural proteins play an important role in the assembly and pathogenicity of virus. VP3 protein is the main structural protein, which is located on the surface of the capsid and is relatively conservative. It is the main determinant of the antigenicity of virus particles and has many antigenic epitopes. DHAV-1 is one of the most widely distributed and harmful duck hepatitis A virus (DHAV-1). In this study, the VP3 of DHAV-1 was expressed and purified in prokaryotic, and rabbit polyclonal antibody against VP3 was prepared. The neutralization activity of VP3 polyantiserum was studied by chicken embryo neutralization test, the possible B antigen epitopes on VP3 were identified by indirect ELISA, and the indirect ELISA method based on VP3 was established to detect DHAV antibody. 1. Prokaryotic expression of DHAV-1 VP3. The expected size of VP3 fragment was obtained by RT-PCR. After T-A was cloned into pMD19-T simpleand subcloned into pGEX-4T-1, the recombinant T plasmid pMD19-VP3 and the recombinant expression plasmid pGEX-VP3. pGEX-VP3 were transformed into BL21DE3, respectively. The recombinant protein expressed at 0.2 mmol / L LIPTGN 37 鈩，

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