【作者】 赵绪永；

【导师】 张改平；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2009， 博士

【摘要】 传染性法氏囊病毒（infectious bursal disease virus,IBDV）是一种无囊膜双链RNA病毒,它所引起的鸡传染性法氏囊病（infectious bursal disease。IBD）是鸡的一种急性高度接触性致死性传染病,是造成养鸡业严重的经济损失的重要疫病之一。病毒受体在病毒吸附、侵入过程中起着极其重要的作用,病毒吸附是启动感染的第一步。研究IBDV的病毒受体,对于探明IBD发生的分子机制,阻断病毒对细胞的吸附和侵染过程,防止传染病的发生有重大意义。鸡胚成纤维细胞（chickenembryo fibroblast,CEF）也是IBDV的易感细胞,通常被用于IBDV的增殖。构建易感细胞的cDNA文库并利用阻断病毒感染的单克隆抗体筛选受体基因是病毒受体研究中最常用的方法。为了研究存在于CEF表面的IBDV受体或感染相关的分子,本研究首先构建了高质量的CEF细胞cDNA T7噬菌体表达文库和真核表达文库,并建立了制备高效电转化感受态细胞E.coli DH10B方法,用于真核表达文库的扩增;然后将生物素-链亲和素（biotin- streptavidin）用于免疫组化,建立了用于筛选阻断病毒感染的单克隆抗体的方法,并制备了阻断IBDV感染CEF的儿株单克隆抗体,为克隆存在于CEF的IBDV受体等感染相关的分子的基因,及揭示IBDV感染CEF的机理奠定了基础。1鸡胚成纤维细胞cDNA T7噬菌体表达文库的构建及鉴定培养CEF细胞,利用oligo（dT）-纤维素亲和层析法制备CEF细胞的mRNA,逆转录合成的cDNA双链,与T7 EcoRⅠ/HindⅢ载体臂定向连接,噬菌体包装,构建cDNA表达文库。测定文库的滴度和并用PCR进行重组子鉴定。将文库扩增并鉴定插入片段的长度及分布。结果显示,文库初始滴度为2.2×10⁷ pfu/mL,扩增后文库的滴度为3.5×10¹⁰pfu/mL。插入片段的平均长度1.3kb,主要分布在0.4～3kb之间。说明成功构建的CEF细胞cDNA T7噬菌体表达文库,为进一步研究CEF细胞以及传染性法氏囊病毒（IBDV）受体研究奠定了基础。2鸡胚成纤维细胞cDNA真核表达文库的构建从CEF提取mRNA,反转录合成双链cDNA,与EcoRⅠ接头连接,经NotⅠ酶切后用spin column除去小于500 bp的片段,连接到预先经EcoRⅠ和NotⅠ双酶切的真核表达质粒pAP3neo载体上,将重组质粒电转化DH10B,测定文库的容量大小并通过colony PCR鉴定插入cDNA片段的大小。经测定,原始文库滴度为2.7×10⁶CFU/mL,重组率大于95%,重组子中插入片段的长度大部分在0.5～2.0kb之间,平均插入外源片段长度大于1.0 kb的约占85.7%。表明构建的文库能够充分满足克隆低丰度mRNA的要求,可以用于筛选IBDV在CEF表面的受体或感染相关分子。3高效电转化感受态细胞的制备为了能够制备高效的电转化感受态细胞,以用于cDNA真核表达文库的转化和扩增。通过对影响转化效率的参数:生长状况、洗液种类、最后细胞密度、电击强度等各个条件进行优化,建立了简单实用的高效电转化感受态细胞E.coli strain DH10B的制备方法,制备的感受态细胞的转化效率达2～6×10⁹cfu/μgDNA,满足了文库转化、基因表达和克隆的特殊需要。同时也为其它大肠杆菌电转化感受态的制备提供了优化思路和借鉴。4阻断IBDV感染CEF的单克隆抗体筛选方法的建立为了制备阻断IBDV感染CEF的单克隆抗体,必须先建立一种有效的筛选单抗的方法。本研究基于免疫细胞化学的方法,采用Biotin-Streptavidin建立了感染阳性细胞数减少实验的方法,可用于阻断IBDV感染CEF的单克隆抗体的筛选。实验表明该方法简单、敏感、实用,是用来筛选阻断IBDV感染的mAb的较好的方法。该方法的建立制备阻断IBDV感染CEF的单克隆抗体奠定了基础,同时该系统的建立也为其它病毒受体的研究从方法学上提供借鉴。5阻断IBDV感染CEF的单克隆抗体的制备和鉴定制备阻断IBDV感染CEF的单克隆抗体,用来筛选或鉴定与单抗作用的受体蛋白或基因,是研究病毒受体的重要方法。CEF是IBDV易感细胞之一,该细胞表面有介导其感染的受体。采取不同方案免疫BALB/c小鼠,经三次免疫后采血测定抗体总效价和阻断感染的效果可见,采用长单层的CEF不加佐剂处理直接腹腔注射免疫的可获得高水平的阻断病毒感染的特异性抗体的产生。用病毒感染细胞数减少实验筛选到3株mAb能阻断IBDV感染CEF,其中一株阻断率达到84.5%。且阻断效率在一定范围内具有剂量依赖性。通过流式细胞仪进行结合实验标明该mAb所针对的表位存在丁CEF细胞表面,但是表达量非常低。推测该蛋白很可能是IBDV的受体之一或重要的感染相关分子,因为不能彻底的阻断感染可见另有其它受体或感染相关分子的存在,这与已报道的文献相符合。该单克隆抗体的制备这为揭示IBDV感染CEF的机理奠定了基础。更多还原

【Abstract】 Infectious bursal disease virus（IBDV）,a member of genus Avibirnavirus of the Birnaviridae family,causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry.Virus receptor plays an important role in virus binding and cell recognition.Viral attachment to a specific receptor on the surface of susceptible host cells is the first step in virus infection.To study the virus infection at the levels of virus binding,receptor identification is critical for understanding the virus-host cell interactions and pathogenesis of the viral disease.Chicken embryo fibroblast（CEF） cells,susceptible host cells of IBDV,commonly are used for the propagation of IBDV. Constructed a cDNA library of a virus susceptible host cell and used a monoclonal antibody（mAb） that can blocks virus infection to screen the library is commonly used in virus receptor identification research.To identify the IBDV receptor or the infection fator on the surface of CEF,we have done series of works as follows.First,a CEF cDNA expression library displayed on phage T7 was constructed via phage display technique,we also constructed a cDNA eukaryotic expression library of CEF and established a simple method to prepare high efficient electrocompetent cells of E.coli strain DH10B.Then on the basis of immunocytochemistry we set up a method in which the biotin-streptavidin systerm is applied.It can be used to screen the monoclonal antibody（mAb） that can blocks IBDV infection.With the mathod we selected three strains of mAb that can block IBDV infection.And we identified some charactors of the mAb.What we have done have settled the basis to clone the gene of IBDV receptor on the surface of CEF or to reveal the mechanism of IBDV infection.1.Construction and quality identification of chicken embryo fibroblast cells（CEF） cDNA expression library displayed on Phage T7To construct the CEF cell eDNA expression library displayed on phage T7 and to identify its quality,mRNA was prepared from CEF by oligo（dT）-cellulose affinity chromatography and reverse transcripted into cDNA.The CEF 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 2.2×10⁷ pfu/mL and 3.5×10¹⁰ pfu/mL.The inserts size largely ranged from 0.4 to 3kb with the average length of 1.3kb.The CEF cell cDNA expression library displayed on phage T7 has high quality.2.Construction and identification of a cDNA eukaryotic expression library of CEF.The mRNA was extracted from CEF and the cDNA was made by reverse transcription. The cDNA was ligated with the EcoR I adaptor and then digested with Not I.Fragments smaller than 500 bp were removed by a spin column.And then the large fragments were ligated into the eukaryotic expression plasmid pAP3neo predigested by EcoR I and Not I. Competent cells DH10B were transformed with the ligated product by electroporation.The size of the cDNA library and that of inserts were also identified through colony PCR.As a result a cDNA eukaryotic expression library of CEF was constructed.The total original bacterial colonies is 8.8×10⁵ in the library.The inserts of the recombinant plasmids larger than 1.0 kb is 85.7%.It indicates that the cDNA library is probably sufficient for cloning of low abundance cDNAs.3.Optimize the protocol of preparing high efficient electrocompetent cellsTo construct a cDNA library,a high efficient electro-competent cell is vital.We optimized several aspects which have a great influence on the transformative efficiency of DH10B electro -competent cell such as OD₆₀₀ value,the final resuspend volume and so on. At last we find out a good method to prepare the electro-competent cell DH10B with the high efficiency of up to 2～6×10⁹cfu/μgDNA.It is satisfied with the harsh requirement of constructing cDNA library,even in gene expression and clone under some special conditions.4.Establish a method to screen the mAb of anti-IBDV receptor on the CEF surfaceAn efficient method must be esbablished which is used for screenning the mAb that can block IBDV infection.In this study based on the method of immunocytochemistry,we employ the Biotin-Streptavidin system to establish an efficient method of the number of infection positive cell reduction test which is used for screenning the mAb that can block IBDV infection.It is a simple and sensitive and practical method for screenning the mAb of blocking virus infection.The method has solve the key problem of screenig the anti-virus receptor monoclonal antibody,which make it possible to reveal the IBDV receptor.It is of great important in methodology because this method can also be used to study to other virus receptor.5.Prepare the mAb of anti-IBDV receptor on the CEF surface and identify some charactors of the mAbThat to prepare the anti-virus receptor monoclonal antibody（mAb） is an efficient method to identify the virus receptor or to clone the receptor gene.Chicken embryo fibroblast（CEF） is a kind of the susceptible cell for infectious bursal disease virus（IBDV）and there are some molecular serves as receptor to mediate the virus infection. Four immunization program was adopted to immunize the BALB/c mouse.With the established mathod we selected three strains of mAb that can block IBDV infection.Some charactors of the mAb were identified.The infection inhibitive ratio of mAb is up to 84.5%. It also has the character of does dependent in infection inhibation.By flow cytometry we definated that the molecular that the mAb against is on the surface of CEF.It indicated that the molecular plays an important role in the course of IBDV infection.We speculate that it is one of the possible IBDV receptors or at least it is a relative element in IBDV infection. Because the mAb can not block IBDV infection completely,we speculate that another receptors or molculars also exsist that play an important role in IBDV infecion.It is consistant with what have been reported that there are at least three receptors existing on CEF.What we have done have settled the basis to reveal the mechanism of IBDV infection.更多还原