【作者】 李铁强；

【导师】 王立群；

【作者基本信息】 东北农业大学 ， 应用微生物学， 2008， 硕士

【摘要】 传染性法氏囊病（IBD）是由传染性法氏囊病病毒（IBDV）引起的急性接触性传染病,是危害世界养禽业的主要传染病之一。IBDV主要侵害雏鸡的中枢免疫器官一法氏囊,导致免疫抑制。1957年本病首次在美国的特拉华州的甘布罗镇（Gumboro）的肉鸡群中发生,20世纪80年代美国又发现了变异株,欧洲国家首先报道有超强毒株（vvIBDV）出现,90年代初vvIBDV传至中国及亚洲其它国家和地区。IBD给养鸡业带来了巨大的经济损失。IBDV的分子生物学研究始于70年代,人们研究发现各IBDV毒株之间的基因变异主要集中在A节段的VP2区域,VP2是血清型特异性抗原,既是IBDV的主要结构蛋白,又是病毒的主要宿主保护性抗原,与病毒中和抗体的诱导、抗原和毒力的变异、细胞凋亡等有关。随着对IBDV的深入研究,人们愈加认识到研究病毒感染机制等基础领域的重要性,其中病毒受体的研究占据着举足轻重的地位,在很多领域都展开广泛研究,尤其人医方面研究更为深入。目前关于IBDV受体蛋白研究的报道很少,虽然有一些相关报道,但仍处于研究病毒自身蛋白与蛋白之间作用的水平,有关病毒蛋白与宿主受体蛋白相互作用的研究还没有报到。酵母双杂交系统是研究蛋白质间相互作用的一种有效方法。该系统能迅速、灵敏地在真核细胞体内检测蛋白生理状态下的互作,并可快速获得目的蛋白的编码基因,近几年来已在很多领域得到应用。本研究利用酵母双杂交系统从CEF cDNA文库中筛选与IBDV弱毒VP2蛋白相互作用的受体蛋白编码基因,并对获得受体蛋白进行初步研究,为研究该病毒的致病机制,防制传染性法氏囊病奠定理论基础。本课题主要研究内容包括以下几方面:1.鸡胚成纤维细胞CDNA表达文库的构建利用gateway技术构建鸡胚成纤维细胞（CEF）文库,以5′端生物素标记的Oligo （dT） primer为引物反转录后连接Adapter,层析柱纯化,通过BP重组反应构建cDNA入门文库,其平均滴度为1.1×10⁶cfu/mL,文库总容量为1.2×107cfu,平均插入片段为2243bp,重组率为100%。通过LR重组反应将入门文库转换为表达文库,经测定平均滴度为5×105cfu/mL,文库总容量为5.5×10⁶cfu,平均插入片段为2411bp,重组率为100%。结果表明,所构建的文库具有较高的重组率和较大的库容量,可作为较高质量的文库来研究IBDV的相关基因,为筛选IBDV在CEF中的细胞受体,研究细胞嗜性提供基础平台。2.IBDV弱毒VP2诱饵载体构建及其自激活作用的检测通过BP和LR两个反应将IBDV Gt株VP2基因克隆到pDEST-32载体上构建诱饵载体,通过酶切和PCR鉴定,并测序正确后,用醋酸锂法转化酵母菌MaV203,在三缺陷板（SC-Trp-Leu-His）上观察其生长情况。结果确定了3AT使用浓度,且证明了诱饵载体本身无自激活活性,为下一步筛选文库提供正确的诱饵载体。3.从CEF文库筛选与IBDV Gt VP2蛋白相互作用受体蛋白及相互作用的验证用构建的诱饵质粒筛选CEF CDNA文库,采用顺序转化的方法,先将Bait质粒转入酵母MaV203中,挑取阳性克隆做酵母感受态细胞,然后再将文库质粒转入含Bait质粒酵母感受态细胞,通过X-gal实验和三种缺陷型培养基筛选,找出19个阳性克隆,提取质粒,测序并比对,结果获得大小两段序列（与原鸡序列相似度为99.9%）,分别为800bp和350bp。最后将两段序列构建成Prey质粒,用同筛库的方法反过来验证与Gt VP2的互作,结果证实了二者的相互作用。4.IBDV VP2蛋白CEF受体的初步研究突变掉CR2₁₁中间所含终止密码子,将其分别克隆到原核载体pET32a和真核表达载体pCAGGS上。经Amp⁺筛选、PCR、酶切和序列分析鉴定表明,插入的位置、大小和读码框均正确。获得重组质粒命名为pET32a-CR2₁₁和pCAGGS-CR2₁₁。将pET32a-CR2₁₁转化宿主菌BL21（DE3）,在IPTG诱导下成功表达了约31kDa的融合蛋白;将构建的pCAGGS-CR2₁₁转染IBDV非允许细胞PK15,做间接免疫荧光实验,结果检测到较强的绿色荧光,说明二者在活体内存在相互作用。更多还原

【Abstract】 Infectious bursal disease virus（IBDV）,the causative agent of infectious bursal disease（IBD）,causes immunosuppression in young chicken by destroying the precursors of B lymphocytes in the bursal of Fabricius. In 1957, the disease first broke out in broilers in Gumboro, Telahua state, America. In the 1980s, the variation strain of IBDV was first appeared in America. Acute IBD caused by very virulent infectious bursal disease virus （vvIBDV） was first reported in Europe lately 1980s. The acute disease was then transmitted into China in the early 1990s, and rapidly spreaded all over Asia and other major parts of the world. The IBDV had caused the significant economic losses in poultry industry for a long time. Through research, people have confirmed that VP2 play a critical role at the induction of NA, mutation of antigen, apoptosis of cell et al., However, there is no reports about receptor protein of IBDV up to now.The development of the yeast two-hybrid system provided a genetic means to identify proteins that physically interact in vivo.The goal of the system is to isolate a protein or proteins that interact with a "bait" protein, this bait protein can be almost any protein from any organism. Over the past ten years, this system and its variants have been extremely useful for detecting and analyzing protein-protein interactions in many fields. One purpose of the research is screening the interactive coding gene with IBDV Gt VP2 from CEF cDNA library by the yeast two-hybrid system. Addition, we take a preliminary research for discoverable gene, all those can as a substantial foundation for us to research pathogenic mechanism of IBDV and prevention of IBD.The following are the main contents of this reseach:1. Construction of chicken embryo fibroblasts cDNA expression library by gateway technology. The mRNA was extracted from chicken embryonic fibroblast. Moreover, single-strand cDNA and double-strand cDNA were synthesized by using biotin-conjugated Oligo （dT） primer in turn. The double-strand cDNA was ligated Adapter and then purified by the cDNA Size Fractionation Columns. After BP recombination reaction, an cDNA entry library was constructed with a titer of 1×10⁶cfu/mL, total clones of 1.2×107 cfu and an average insertion size of about 2243bp. After LR recombination reaction, the cDNA entry library was transformed into expression library which took on a titer of 5×105cfu/mL, total clones of 5.5×10⁶ cfu and an average insertion size of about 2411bp. The results indicate that the constructed cDNA expression library performs a remarkable high value in both recombination rate and library coverage. As a result, the cDNA expression library, with its good quality, may facilitate to identify the receptors associated with the resistance against IBDV in chicken embryonic fibroblast and to cast new light on the mechanism of cellular tropism. Moreover, it may also provide data of chicken embryonic fibroblast in transcription level and may be helpful to study its biological functions.2. Through BP and LR reaction, the fragments of infectious bursal disease virus （IBDV） Gt VP2 was cloned into pDEST-32 vector by Gateway technology, after confirmation with restriction enzyme digestion, PCR and sequence analysis, the plasmids were transformed into the yeast cell MaV203 with ployethylene glycol/lithiuma acetate method, then the growths of transformation were observed on the SC-Trp-Leu-His selective plate, to detect the self-activation of bait vectors. At last, determination 3AT density and verification the bait vector neither the ability of outonomous reporter gene activation, nor toxicity to the yeast host-cells.3. Transform bait plasmid into yeast competent cell, then to select the positive clone as the yeast competent cell for CEF library plasmid. Through X-gal test and three selective medium, we found 19 postive clones, extraction the plasmid and sequence. In the result, we obtain two gene segments （similar Gallus gallus gene as 99.9%）, they are 800bp and 350bp respectively. Continue, construction prey plasmid with two gene segments, to test the interaction between two segments with Gt VP2 again.4. Delete the termination codon by PCR technology, then little segment (CR2₁₁) was cloned into expressing vector pET32a and vector pCAGGS, the recombinants were transformed into E.coli DH5α. The recombinant plasmid was identified by restriction enzyme digestion, PCR and sequence analysis and named pET32a-CR2₁₁ and pCAGGS-CR2₁₁. The recombinant plasmid (pET32a-CR2₁₁) was transformed into E.coli BL21（DE3） and induced by IPTG. SDS-PAGE results indicated that the fusion protein was about 31kDa. The pCAGGS -CR2₁₁ was transfected into PK15 cell, IFA showed that there are strong green fluorescence on the cell hole （PK15）, It proved the interactive role between Gt VP2 and CR2₁₁ in vivo.更多还原