【作者】 刘华兰；

【导师】 郭爱珍；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 干扰素（interferon,IFN）,是一类具有广谱抗病毒、抗肿瘤和增强免疫功能的细胞因子。自1957年Isaacs和Lindenmann首先发现干扰素以来,人们对IFN的研究、开发及应用从未停止过,目前IFN已被广泛应用于人医临床多种疾病的治疗。1980年WHO根据IFN抗原特异性的不同将其分为三类:IFN-α、IFN-β、IFN-γ;后来又依IFN作用的受体不同而分为两型:Ⅰ型IFN和Ⅱ型IFN,Ⅰ型包括IFN-α、β、ω、τ、δ等;Ⅱ型只有IFN-γ。相对而言,兽医界对动物干扰素的研究起步较晚。由病毒、细菌等病原微生物侵染动物所引起的各种传染性疾病严重制约了各个国家和地区养殖业的健康发展,同时也是人类健康的巨大隐患。因此,研究动物干扰素对动物传染性疾病的防治具有重大的经济价值和社会意义。国内外奶牛养殖场在奶牛养殖过程中,常面临多种疾病,例如:奶牛乳腺炎、口蹄疫、牛结核病等。疾病不但严重影响奶牛的产奶量和牛奶的质量,而且给奶牛场造成严重的经济损失。干扰素是细胞介导免疫的重要免疫分子,目前国内外学者利用基因工程技术研制牛干扰素γ及大量制备γ干扰素已取得了很多成果,而对IFN-α、IFN-β的基因工程技术制备还未见报道。近年来研究表明,干扰素β（IFNβ）与干扰素α（IFNα）一样,是一种具有多种生物学作用的重要细胞因子,虽然其与IFNα抗病毒作用机理相似,但IFNβ具有独特性,而这一独特性与IFNβ的抗病毒效果紧密相关。鸡传染性支气管炎（Avian Infectious Bronchitis,IB）是由鸡传染性支气管炎病毒（Infectious Bronchitis Virus,IBV）引起的一种高度接触性的病毒性传染病。主要引起鸡的呼吸系统疾病、肾炎并伴随产蛋量和蛋品质的下降。本实验室陈汉阳博士对IBV的天然受体进行了探索性研究,通过将gAPN转染IBV的非敏感细胞(（PK-15,HeLa）,构建真核表达体系。通过间接免疫荧光、流式细胞术、半定量RT-PCR等试验证实IBV可以在这些转染后细胞上增殖。首次利用转基因细胞证明gAPN是IBV的天然受体之一（陈汉阳,2006）。为了进一步验证gAPN为IBV的天然受体,进行本动物实验验证是十分必要的。鉴于以上研究背景,本研究主要开展了一下内容:1.BoIFN-β基因的克隆、测序分析及其原核表达以植物血凝素（PHA）诱导的牛外周血淋巴细胞中提取的总RNA为模板,采用RT-PCR方法克隆扩增出编码牛干扰素β成熟蛋白基因（mBoIFNβ,498bp）。经测序结果证实扩增得到的mBoIFNβ与Genbank上所报道的牛干扰素β基因（E00137）的同源性为100%;牛β干扰素与野猪、马、猫、狗、人、鼠的氨基酸序列同源性分别为59%、50%、51%、46%、49%～51%、36%～43%。将mBoIFNβ插入pET-28a（+）表达载体,并转化宿主菌BL₂₁,经IPTG诱导后,得到高效表达。表达产物经SDS-PAGE分析,证明BoIFNβ能够被诱导并高效表达,主要形成不溶性包涵体,大小约为28.6kDa。通过对宿主菌不同时间的诱导发现目的蛋白在诱导后4小时的表达量最高。2.BoIFN-β的真核表达载体的构建为了获得高效分泌表达的重组牛β干扰素,利用基因工程技术,将编码牛β干扰素成熟蛋白基因（mBoIFNβ,495bp）,亚克隆到含分泌信号肤序列的毕赤酵母表达载体pPIC9K中,构建成分泌型重组表达载体pPIC9K-mBoIFNβ。用化学方法（LiCl）将线性化的mBoIFNβ与ssDNA共转化入毕赤酵母菌株GS115,转化子经MD平板筛选和PCR鉴定后,得到的阳性菌株,再以高浓度的G418筛选到多拷贝重组子。3.gAPN在IBV自然宿主鸡体内的表达研究为了进一步验证gAPN作为IBV的天然受体之一,本试验以鸡为实验动物,利用RT-PCR技术,分别检测了gAPN基因在鸡组织的转录水平。同时用免疫组化技术,检测了gAPN在不同鸡组织的表达。结果证实,gAPN基因在各组织器官的转录由高至低的顺序为:卵巢、输卵管、气管、脾、回肠、肺、肝、十二指肠、肾和心脏,与IBV的组织嗜性相一致。免疫组化证实,gAPN在小肠（哪一段?）、肾、肝、心、脾等具有不同程度的表达。此外,还用流式细胞术检测了鸡胚细胞表达gAPN的水平及与IBV的结合能力。更多还原

【Abstract】 Interferon(IFN) is a kind of cytokine with antiviral,anti-tumor and regulatory immunity functions.Since Isaacs and Lindenmann reported in 1957,the studies on IFN and its development and application never stopped.Now it has been utilized in the control of variable diseases for human beings.In 1980,IFN was classified into three categories,IFN-α,IFN-βand IFN-γ,by world health organization based on the antigenic specificity.Afterwards it was classified into two types on their different acceptors,typeⅠIFN and typeⅡIFN.TypeⅠIFN includes IFN-α,β,ω,τ,δetc,and typeⅡIFN only contains IFN-γ.Research about IFN-γis very hot during last decades and several products about recombinant IFN-γhave been applied in market.However,research on IFN-α,and IFN-βis not as much as that of IFN-γ.The IFN-βproducts for different specieses are very rare. Latest research showes that IFN-βpossesses multiple functions as one of the important cytokines.Although anti-virus mechanism is similar to IFN-α,IFN-βhas some special characters which are highly related to its anti-virus effect.The studies on the animal IFN began comparatively later in veterinary medicine.For cattle,various infectious diseases such as mastitis,foot-and-mouth disease, bovine tuberculosis,etc,caused by viral or bacterial pathogens not only adversely affect the development of national and regional cattle industries,but also endanger human health in the whole world.The research on IFN-βwill improve the disease control of cows by enhancing cow’s immunity and establishing a defense mechanism against viruses and bacteria.Avian Infectious Bronchitis(IB) is a highly contagious viral infection of the domestic fowl caused by infectious bronchitis virus(IBV).It can lead to respiratory disease,kidney inflammation with low production and poor quality of eggs.The previous study in this lab has lauched some initial research on identification of the IBV natural receptor,HeLa and PK-15 cell monolayers that do not permit natural infection by IBV were transfected with gAPN-neo plasmid.The transfected cells became to be permissive to IBV.However,receptor function of gAPN in the natural host chicken was not studied. Thus,part of this dissertation continued to determine the expression of gAPN in chicken tissue and the interaction between IBV and the tissues.The main results were summarized as follows:1.Cloning and sequence analysis of bovine interferon-beta gene and its expression in E.coliThe total RNA was extracted from peripheral blood mononuclear cells(PBMC) which was isolated from bovine and induced with phytohemagglutinin(PHA),then the mature peptide of bovine interferon beta(mBoIFN-β,498bp) was amplified by RT-PCR. The result of sequencing demonstrated that the cloned mBoIFN-βgene had 100% homology at nucleotide level to that published on Genbank,Furthermore,the mBoIFN-βhad 59%、50%、51%、46%、49%～51%、36%～43%homology at amino acid level respectively to that of Sus scrofa,horse,feline,canis,human,and mice.The mBoIFN-βgene was inserted into pET-28a(+) and the recombinant plasmid was transformed into BL21 E.coli strain,and the expression was induced by IPTG.SDS-PAGE demonstrated that the protein with the size of 28.6 kDa) mainly existed in insolvable inclusion body. The best inducing time was 4 h after the addition of IPTG..2.Construction of eukaryotic expression vector of IFN-βin pichia pastorisTo highly express secreted bovine interferon-beta,the signal peptide was excised from the BoIFN-βgenes and the mature peptide(mBoIFN-β,495 bp) was cloned into the yeast-Escherichia shuttle vector pPIC 9K to construct secreting recombinant expression plasmid of pPIC 9K- mBoIFN-β.The pPIC 9K-mBoIFN-βwas linearized by SalⅠand co-transformed with ssDNA into Pichia pastoris cells GS115(defective with histidine) with LiCl.The transformants were selected with MD culture plates and the mBoIFN-βgene insertion was identified by PCR.The multicopy recombinant Pichia pastoris strain was selected by G418 resistance.3.The expression of gAPN in chicken,the IBV natural host In order to demonstrate the receptor function of gAPN in IBV natural host,this study used the semi-quantity RT-PCR to analyze the levels of gAPN gene transcription in chicken tissues.Meanwhile,the immuno-histochemistry was performed to confirm the expression of gAPN in chicken tissues.As a result,the tissues with gAPN gene transcription in descending order was as follows:ovary,oviduct,trachea,spleen,ileum,lung,liver,duodenum, kidney,jejunum,and heart.This distribution is in agreement with the tissue tropism of IBV.the immuno-histochemistry demonstrated that gAPN was expressed in ileum,? kidney,liver、heart、trachea.Besides,flow cytometry showed that chicken embryo cells expressed gAPN and was able to attach IBV.更多还原