【作者】 张红；

【导师】 张改平；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2007， 博士

【摘要】 传染性法氏囊病毒（infectious bursal disease virus,IBDV）是引起鸡传染性法氏囊病（infectious bursal disease,IBD）的致病病原体,主要侵害幼龄雏鸡中枢免疫器官-法氏囊,损伤B淋巴细胞,引起严重的免疫抑制。雏鸡易感染其它传染病和疫苗接种失败,对养鸡业造成重大损失。病毒受体是公认的引发病毒感染宿主细胞的主要决定因素。吸附是启动病毒感染的第一步,也是决定病毒感染能否成功的关键环节,细胞受体在病毒吸附及感染过程中扮演了极其重要的角色,是感染发生的关键因子。为了揭示IBDV B细胞受体的特性及其功能,本论文采用T7噬菌体展示技术构建高质量的呈现在T7噬菌体的鸡B细胞cDNA表达文库,通过生物淘选过程筛选与IBDV高度亲和的受体蛋白,利用基因重组技术实现了备选受体分子在非易感细胞COS7细胞膜的定位表达。病毒结合试验和竞争/抑制试验证实膜定位表达鸡Igλ轻链的COS7细胞与病毒特异性结合。研究结果表明鸡Igλ轻链是病毒位于B细胞上重要的结合受体。为从分子水平深刻理解病毒与宿主细胞的相互关系以及病毒吸附和感染易感B淋巴细胞的机制提供了重要理论依据。1构建鸡B淋巴细胞cDNA T7噬菌体表达文库并对文库质量进行鉴定利用oligo（dT）-纤维素亲和层析从SPF雏鸡法氏囊细胞制备mRNA,合成的双链cDNA定向克隆于T7 EcoRⅠ/HindⅢ载体臂,包装并构建cDNA表达文库。对文库进行滴度测定和重组子测定。扩增文库并进行PCR鉴定插入序列的长度及分布。结果显示文库初始滴度为5×10⁷pfu/mL,扩增后滴度达到1.88x10¹⁰pfu/mL。插入片段平均长度1.44kb,主要分布在0.75-2kb之间。表明所构建的鸡B淋巴细胞cDNA T7噬菌体表达文库的质量能充分满足从文库筛选受体基因的需要。2传染性法氏囊病毒对鸡B细胞T7噬菌体表达文库的亲和筛选以纯化的IBDV病毒为筛选配体,对构建的鸡B细胞cDNA噬菌体表达文库共进行了4轮亲和筛选。每轮筛选后对洗脱的噬菌体进行噬斑计数计算噬菌体滴度,并将各轮投入/产出比进行比较,分析富集效果。投入/产出比分析表明,每一轮淘选均有较好的富集率。四轮的噬菌体滴度分别为:3.8×10²pfu/mL、2.4×10⁴pfu/mL、2.7×10⁶pfu/mL、3.2×10⁶pfu/mL:回收率分别为2.6×10^-6、4.2×10^-4、3.7×10^-2、3.1×10^-2;对筛选的噬菌体运用噬菌斑印迹（plaque lift）和phage-ELISA试验鉴定其与病毒结合的特异性,获得与病毒高度特异性结合的噬菌体克隆。随机挑选80个阳性噬菌体克隆进行DNA测序和序列分析,显示有70%的噬菌体插入序列与鸡Igλ轻链有高度同源性,核苷酸和氨基酸序列同源性分别达到96%和92%。3鸡Igλ轻链跨膜嵌合分子真核表达载体构建及在COS7细胞表达将扩增的鸡Igλ轻链基因与牛IgGFc受体γRⅡ跨膜区（R2T）通过融合PCR技术形成嵌合跨膜分子λR2T,定向克隆于真核表达载体pcDNA3,利用γRⅡ跨膜区对细胞膜的锚定功能以及鸡Igλ轻链信号肽序列的引导作用实现重组Igλ轻链在COS7细胞膜的定位表达,在IBDV非易感细胞-COS7上完成备选受体蛋白的重建。为了确定嵌合跨膜分子在细胞膜上的成功表达,巧妙运用一种“特洛伊木马”策略将鸡Igλ轻链信号肽序列插入到增强型绿色荧光蛋白真核表达载体pEGFP-C1的EGFP编码序列的起始密码子后,构建带信号肽的中间载体pEGFP-C1-SP,将嵌合跨膜分子λR2T克隆于中间载体的多克隆位点,转染COS7细胞,荧光显微镜观察到重组鸡Igλ/GFP融合跨膜蛋白在COS7细胞膜的表达。将鸡Igλ轻链基因分别克隆于pcDNA3载体和pEGFP-C1-SP中间载体,构建分泌表达的重组载体并实现重组Igλ在COS7细胞的分泌表达。4鸡Igλ轻链作为病毒B细胞受体的生物学功能鉴定COS7细胞分泌表达的鸡重组Igλ蛋白经Ni-NTA spin column亲合柱层析纯化,VOPBA试验结果证明分泌表达的鸡重组Igk蛋白能与法氏囊病毒特异性结合。表面表达有鸡Igλ轻链的COS7细胞与病毒进行结合试验,流式细胞术分析结果显示其阳性细胞比例（结合了病毒的细胞）为83%,细胞平均荧光强度（mean fluorescence intensity,M.F.I）远远高于空质粒转染的COS7细胞。重组鸡Igλ轻链或是抗Igλ单抗均能够显著抑制病毒与COS7细胞和B细胞的结合,其抑制作用呈现剂量依赖性特征。感染试验结果则显示鸡Igλ轻链不能介导病毒对细胞的感染。研究结果证实鸡Igλ轻链是病毒重要的结合受体,病毒借助与鸡Igλ轻链的结合从而特异性吸附于B细胞表面,使病毒集聚于细胞,为侵入细胞和引起感染迈出了关键的一步。更多还原

【Abstract】 Infectious bursal disease（IBD）is a viral disease of young chickens characterized by necrosis and depletion of lymphoid tissues,especially the bursa of Fabricius.The causative agent of this disease,IBD virus（IBDV）is a member of the genus Avibirnavirus of the family Birnaviridae which consists of double-stranded RNA genome and responsible for severe immunosuppressive disease that causes significant losses to the poultry industry.As with all viruses,IBDV needs to penetrate target cells to cause infection by a communication of the receptor binding.The attachment of the virus to a specific receptor on the surface of susceptible host cells is the first step in virus infection and has attracted attention as targets for infection prevention.It is of importance to study the virus infection at the molecular level of virus binding for understanding the virus-host cell interaction and pathogenesis of IBD.However,the investigation on the complex biochemical processes involving virus binding to host cell surface molecules is hindered by the lack of knowledge on the identities of the virus receptor of IBDV.To identify the receptor which may determine the susceptible B lymphocyte binding and viral infection,a chicken B cell cDNA expression library displayed on phage T7 was constructed via phage display technique.Chicken Igλlight chain was identified after the library secreening and DNA sequencing.Virus binding and inhibition assay confirmed that the IBDV-nonpermissive COS7 cells bound viruses specifically once the candidate receptor was expressed on the cell membrane of COS7.Data suggest that chicken Igλlight chain is a binding receptor for IBDV on B cells and facilitate virus binding to B cells.1 Construction and quality identification of chicken B cell cDNA expression library displayed on Phage T7To construct the chicken B cell cDNA expression library displayed on phage T7 and to identify its quality,mRNA was prepared from bursa of Fabricius in a SPF chicken by oligo（dT）-cellulose affinity chromatography and reverse transcripted into cDNA.A chicken B cell cDNA expression library was constructed after cloning cDNA into T7 EcoRI/HindⅢvector arms and in Vitro packaging.Titer and recombinant rate of the prime library were determined.The size of the inserts was identified by PCR after a round of library amplification.The titer of the prime constructed library and amplified library was 5×10⁷ pfu/mL and 1.88×10¹⁰pfu/mL.The inserts size largely ranged from 0.75 to 2kb with the average length of 1.44kb.The chicken B cell cDNA expression library displayed on phage T7 has high quality.2 Screening the T7 expression library for phage clones with high affinity for IBDVPurified IBDV was used as the bait in a biopanning procedure.Immobilized IBDV was probed to bind T7 phages that were expressed proteins from a chicken B cell cDNA library.The phage recovery percentage was assessed by output/input phages as a measure of enrichment and determined rounds of biopanning.After four rounds of biopanning,the bound phages were isolated and their affinity and specificity for IBDV was detected by plaque lift and phage ELISA.80 candidate clones were sequenced and analyzed.Most sequences of the clones containing that may be involved in IBDV binding to B cells were chicken Ig lambda light chain sequences from open reading frame of the protein.The sequences show high identities to chicken Ig lambda light chain in GeneBank.3 Expression of recombinant chicken Igλlight chain in virus nonpermissive COS7 cellsRecombinant DNA ecoding chimeric protein incorporating transmembrane region of bovine IgG Fc receptorγRⅡ（bFcRγRⅡ）and chicken Igλlight chain was generated by fusion PCR.pEGFP-C1-SP,the vector with signal sequence was then created by fusing signal peptide of chicken Igλto GFP N terminus.The chimeric full length sequence was respectively cloned into modified pEGFP-C1-SP and pcDNA3 plasmid using double restriction sites.Mammalian expression vector pEGFP-C1-SP-λR2T and pcDNA-λR2T ware successfuLly constructed.The chimeric transmembrane protein was located principally on the plasma membrane in COS7 cells transfected with vector pEGFP-C1-SP-λR2T under fluorescent microscope or with vector pcDNA-λR2T by flow cytometric analysis.The secretable expression of chicken Igλwas observed under fluorescent microscope and in Western blotting by inserting the chicken Igλgene into eukaryotic expression plasmid pEGFP-C1-SP and pcDNA3 and then transfecting the recombinant vector into COS7 cells via lipofectamin.4 Biological function of chicken Igλlight chain as a cellular receptor for IBDV recombinant vector into COS7 cells via lipofectamin.4 Biological function of chicken Igλlight chain as a cellular receptor for IBDVTo examine whether the recombinant chicken Igλcould interact with IBDV,VOPBA was performed.The purified IBDV bound to recombinant chicken Igλwas detected with MAb against IBDV nucleocapsid protein,while no band was observed in negative controlTo determine function of chicken Igλlight chain as a IBDV receptor that can facilitate IBDV binding to nonpermissive cells,COS7 were transfected with plasmid constructs coding for chimeric transmembrane protein and exposed to virus on ice to allow virus binding.COS7 cells transfected with chimeric construct showed high levels of virus binding in comparison to nontransfected COS7 cells.The binding of IBDV occurred in a dosed-dependent manner.COS7 cells without expression of chicken Ig lambda light chain and lymphocytes from the bursa of Fabricius of 4-week-old chicken were exposed to virus,based either on immunofluorescence microscopy or flow cytometry using a anti-IBDV antibody.BF lymphocytes showed high levels of virus binding,in contrast,very little binding was observed with COS7 cell control.For competition of IBDV binding the viruses were incubated with recombinant chicken Igλlight chain or antiserum specific for IBDV before incubating with cells. Inhibition of virus binding was obtained by incubation of cells with mAb against chicken Igλlight chain before being tested for the ability to bind viruses.Detection of bound viruses was performed by flow cytometry.The inhibition effects on virus binding were much more obvious than that in control.These results demonstrated that chicken Igλlight chain may act as a cellular receptor facilitating IBDV binding to chicken B lymphocytes. The findings in this study provide a basis for further exploration of molecular mechanism by which IBDV infects chicken B lymphocytes.更多还原