鸭坦布苏病毒E蛋白中和表位的鉴定和受体的筛选

发布时间：2018-03-29 21:07

本文选题：DTMUV　切入点：E　出处：《华中农业大学》2017年硕士论文

【摘要】：鸭坦布苏病毒(Duck Tembusu virus,DTMUV)是2010年初在我国的江浙地区新发的一种黄病毒,引起蛋鸭的产蛋量急剧下降甚至停止,给我国的养殖业造成严重的经济损失。近些年对其分离鉴定、基因组特征和诊断方法的报道较多,但是对于该病毒中和表位和受体的研究成果较少,尤其是针对DTMUV蛋白受体的研究至今仍无进展。囊膜E蛋白作为DTMUV主要的结构蛋白,不仅可以刺激机体产生中和抗体,同时与受体结合介导病毒的入侵,对揭示病毒感染机制和免疫反应至关重要。本研究克隆并表达了DTMUV E蛋白,制备了多株单克隆抗体,筛选出一株具有中和活性的单克隆抗体,通过鉴定发现其中和表位位于E蛋白的Domain I区域,这是首次在黄病毒E蛋白Domain I区域发现的中和表位,丰富了我们对黄病毒中和表位的认识。通过对中和抗体的可变区进行克隆、序列分析和单链抗体(Sc Fv)的重组质粒构建,发现其轻链互补决定区序列具有显著的特异性,纯化的单链抗体具有一定的抗病毒效果,为针对该病的预防与治疗奠定了基础。采用免疫共沉淀和病毒-受体蛋白结合试验(VOPBA)技术对DTMUV的受体进行了初步筛选,但是未检测分离到与DTMUV易感细胞结合的蛋白受体,通过DTMUV E蛋白与血红细胞的血凝试验检测发现E蛋白具有一定的血凝活性,提示该病毒受体可能属于糖类,为该病的防控提供新的思路。具体研究内容如下:1.DTMUV E单克隆抗体的制备与鉴定本研究根据实验室分离的鸭坦布苏病毒WH2014株E基因序列(Gen Bank KY235382),构建了去信号肽成熟的E蛋白的重组质粒p ET-28a-E,采用原核诱导表达和纯化系统获得高纯度的E蛋白。将纯化后的E蛋白免疫BALB/c小鼠制备针对E蛋白的多株单克隆抗体,通过间接ELISA、间接免疫荧光和Western blot筛选获得2株稳定分泌抗体的杂交瘤细胞株,分别命名为3B8G和3F12G。其腹水间接ELISA效价分别为1:51200和1:409600;亚类鉴定结果显示,Mc Ab 3B8G重链为Ig G1类,Mc Ab 3F12G重链为Ig G3类,轻链均为κ链;间接免疫荧光和Western blot结果显示,两株单抗均能与过表达的E蛋白具有特异性结合反应,并且与DTMUV同样产生特异性反应。病毒中和实验结果表明,Mc Ab 3B8G对DTMUV的中和效果较好。2.E蛋白中和抗原表位的鉴定通过生物信息学方法分析DTMUV E蛋白空间构象,对E蛋白的三个结构域进行截短突变体真核表达重组质粒的构建,间接ELISA、间接免疫荧光和Western blot检测发现Mc Ab 3B8G识别E蛋白的DEI区域,Mc Ab 3F12G识别E蛋白的Domain II区域。为了进一步鉴定效果较好的中和抗体3B8G在E蛋白DEI区域内的抗原表位最小功能区,本研究通过构建一系列覆盖E蛋白Domain I全长的截短突变体真核表达重组质粒,经IFA和Western blot检测,精确扫描定位了Mc Ab 3B8G识别的一个B细胞线性表位1FSCLGMQ7。3.抗DTMUV E蛋白单链抗体的制备与鉴定本研究在成功制备稳定分泌抗DTMUV E蛋白单抗杂交瘤细胞株的基础上,对两株单抗的VL区域扩增,比对分析其序列发现两株抗体在VL区域相似性较低,决定了抗体识别E蛋白结构域的特异性。通过基因工程方法,拼接抗DTMUV E蛋白单链抗体基因并构建了重组表达载体p ET 28a-DTMUV-E Sc Fv,表达纯化得到的单链抗体具有良好的抗病毒活性。4.DTMUV E蛋白受体的筛选为了研究DTMUV在易感细胞上的受体蛋白分子,本研究表达纯化了E蛋白和Domain III蛋白,通过间接免疫荧光法筛选与DTMUV易感细胞稳定结合的蛋白,发现E蛋白能够稳定结合DEF细胞、DF1细胞和Vero细胞。然后采用膜蛋白提取试剂盒提取以上三种细胞的膜蛋白,将膜蛋白、纯化的E蛋白和抗DTMUV的单抗进行免疫共沉淀和病毒-蛋白结合筛选,结果均未筛选到特异性可分离的DTMUV蛋白受体,血凝试验提示鸭坦布苏病毒的受体可能为糖类,为进一步研究病毒受体提供了思路。综上所述,本研究鉴定出DTMUV位于E蛋白Domain I结构域内的中和表位;分析了两株识别不同抗原区的单克隆抗体轻链可变区序列差异,构建了抗DTMUV E蛋白的单链抗体融合表达载体;对DTMUV的蛋白受体分子进行了初步筛选。本研究对建立鸭坦布苏病毒的防控与治疗技术具有重要意义。
[Abstract]:Duck Tembusu virus (Duck Tembusu, virus, DTMUV) is a kind of flavivirus in early 2010 China's Jiangsu and Zhejiang new hair, caused by the sharp decline in duck egg production or even stop, causing serious economic losses to the aquaculture of our country in recent years. The isolation and identification, characteristics and diagnostic methods of the genome are reported, but research on the virus neutralizing epitopes and receptor are few, especially on DTMUV receptor is still no progress. Envelope E protein as the major structural protein of DTMUV, not only can stimulate the organism to produce neutralizing antibody, combined with virus and receptor mediated invasion, to reveal the mechanism of viral infection and the immune response is crucial the study on cloning and expression of DTMUV E protein, polyclonal monoclonal antibodies were prepared and screened a strain of monoclonal antibodies with neutralizing activity, through the identification of the epitopes found a In the Domain I region of E protein, which was first discovered in the Domain I region of flavivirus E protein epitopes, to enrich our understanding of virus neutralizing epitopes were cloned. Through variable region of neutralizing antibody, and sequence analysis of scFv (Sc Fv) to construct the recombinant plasmid sequence has found out. The remarkable specificity of light chain complementarity, the purified scFv has certain antiviral effect, for the prevention and treatment of the disease of the foundation. By immunoprecipitation and virus - receptor protein binding assay (VOPBA) technique on DTMUV by the initial screening, but not easy to separation and DTMUV detection sense of cell binding protein receptor, by hemagglutination test for the detection of DTMUV E protein and red blood cells showed that E protein has hemagglutination activity, suggesting that the virus receptor may belong to carbohydrate, provide for disease prevention and control of new Ideas. The specific contents are as follows: preparation and identification on the basis of duck Tembusu virus WH2014 strain E gene sequence of 1.DTMUV E isolated monoclonal antibody (Gen Bank KY235382), to construct the signal peptide of mature E protein recombinant plasmid P ET-28a-E, induced by prokaryotic expression and purification system of high purity the E mice were immunized with BALB/c protein. The purified E protein to prepare monoclonal antibodies to E protein, indirect ELISA, indirect immunofluorescence and Western blot screened 2 strains of stable antibody secreting hybridoma cell lines, named 3B8G and 3F12G. in the ascites titer of ELISA were 1:51200 and 1:409600; subtype identification showed that Mc Ab 3B8G heavy chain Ig G1, Mc Ab 3F12G heavy chain Ig G3, were light chain kappa chain; indirect immunofluorescence and Western blot results showed that the two McAbs with overexpression of E The protein has a specific binding reaction, and DTMUV also produced specific virus neutralizing reaction. The experimental results show that the identification of Mc Ab 3B8G of DTMUV and.2.E protein and better antigen through bioinformatics analysis of DTMUV on the conformation of E protein, E egg three domain of truncated mutant really nuclear construction, expression recombinant plasmid of indirect ELISA, immunofluorescence and Western blot detected DEI Mc Ab 3B8G E region recognition protein, Domain II Mc Ab 3F12G E protein region recognition. In order to further identification of neutralizing antibody 3B8G good effect in E protein DEI region epitope minimum functional areas, this study through the construction of a series of E Domain I protein covering full-length truncated mutant recombinant eukaryotic expression plasmid, the IFA and Western blot detection, Mc Ab 3B8G scanning and positioning accurate identification of a B cell Preparation and identification of the research in the successful preparation of E protein stably secreting monoclonal antibody against DTMUV linear epitopes on 1FSCLGMQ7.3. protein of single chain antibody against DTMUV E, VL region of two strains of monoclonal antibody amplification, the sequence alignment analysis showed that two strains of antibodies in the VL region of low similarity, determines the specific antibody recognition of E protein domains. By gene engineering method, protein splicing of single chain antibody against DTMUV E gene and construct the recombinant expression vector p ET 28a-DTMUV-E Sc Fv, the expression of purified scFv obtained with antiviral activity of E protein.4.DTMUV receptor of DTMUV in order to study the receptor protein in susceptible cells. The study on the expression and purification of E protein and Domain III protein by indirect immunofluorescence assay and screening of DTMUV susceptible cell stable binding protein, E protein can stably bind to DEF cells, DF1 Cell membrane protein and Vero cells. Then the membrane protein extraction kit to extract more than three kinds of cells, the membrane protein, the purified E protein and anti DTMUV monoclonal antibody immunoprecipitation and virus protein binding screening, the results were not screened for specific separation of DTMUV protein by hemagglutination test indicated that the body. Duck Tembusu virus receptor may be sugar, provides ideas for further study of virus receptor. In conclusion, this study identified DTMUV in E protein Domain I domain in neutralizing epitope; analysis of antibody light chain variable region sequence difference of two strains of monoclonal antigen recognition in different areas, construction of anti DTMUV scFv antibody E fusion protein expression vector; protein receptor molecules on DTMUV were screened. Has important significance for prevention and treatment technology for the establishment of duck Tembusu virus in this study.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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