【作者】 吕平；

【导师】 伍晓雄；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2008， 硕士

【摘要】 干扰素（Interferon,IFN）是一种具有抗病毒,影响细胞生长、分化和免疫调节等功能的蛋白质,在兽医临床上,干扰素在对疾病的预防、治疗以及疫苗佐剂的应用等方面发挥着重要作用。目前,IFN已广泛应用于人医临床多种疾病的治疗。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,同时也对人类健康存在着巨大危害。因此,动物干扰素的研究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。本研究以猪干扰素α（PoIFN-α）、猪干扰素β（PoIFN-β）与猪干扰素γ（PoIFN-γ）为研究对象,构建了pcDNA3.1-EGFP-PoIFN-α/β/γ（命名为pcDNA3.1-IFN-α/β/γ）和rAAV-PoIFN-α/β/γ（命名为AAV-PoIFN-α/γ）真核表达载体,并转染哺乳动物细胞293T,Western-blot检测到了293T细胞中干扰素蛋白。主要研究内容如下:1.pcDNA3.1-IFN质粒构建和制备根据本实验室已扩增到的PoIFN-α/β/γ全长基因设计引物,在PoIFN-α/β/γ上下游引入NheⅠ和HindⅢ酶切位点,以PoIFN-α/β/γ全长基因为模板,PCR扩增得到PoIFN-α/β/γ基因,将该基因亚克隆到pcDNA3.1-IFN载体上,转化大肠杆菌DH5α,挑选单菌落,LB培养基培养,菌液PCR鉴定,经测序鉴定获得正确pcDNA3.1-PoIFNd质粒,扩大培养,大量提取质粒并纯化。2.rAAV-IFN质粒构建和制备根据本实验室已扩增到的PoIFN-α/β/γ全长基因设计引物,使得PoIFN-α/β/γ上下游引入EcoRⅠ和HindⅢ酶切位点,以PoIFN-α/β/γ全长基因为模板,PCR扩增得到PoIFN-α/β/γ基因,将该基因亚克隆到AAV载体上,转化大肠杆菌DH5α,挑选单菌落,LB培养基培养,菌液PCR鉴定,证明获得正确AAV-PoIFN质粒,扩大培养,大量提取质粒并纯化。3.猪干扰素在293T细胞中的表达腺相关病毒载体三质粒系统（AAV-PoIFN、AAV-RC、AAV-pHelper,按1:1:1的比例混合）,pcDNA3.1-PoIFN,lipofectamine^TM2000脂质体转染293T细胞,Western-blot检测了培养液上清中干扰素蛋白。更多还原

【Abstract】 IFN（Interferon,IFN） is a kind of anti-virus,regulating cell growth and differentiation,immune-modulated protein.In veterinary medicine,it is important to prevent disease and cure virus-disease,to study vaccine adjuvant et al.Relatively speaking,the studies on the animal IFN begin comparatively late in veterinary medical circles.In pork-producing community,various infectious diseases which are caused by viral or bacterial pathogens,badly restricted the developmengt of natural and regional farming industries,and also seriously affect human health in whole world.hence,it’s necessary and great meaningful in both economic development and societal stabilization to prevent and control infectious diseases.In light of the consideration,in this study porcine interferon-alpha（PoIFN-α）,porcine interferon-beta （PoIFN-β） and porcine interferon-gamma（PoIFN-γ） were investigated.The PoIFNs were respectively subcloned into pcDNA3.1-EGFP and rAAV and constructed pcDNA3.1-EGFP-PoIFN-α/β/γ（named pcDNA3.1-IFN-α/β/γ）和rAAV-PoIFN-α/β/γ（named AAV-PoIFN-α/β/γ） then six eukaryotic expression plasmids respectively transfected into 293T cell,The culture supernatants were detected with SDS-PAGE.The main researches included as below:1.Three pairs primmer were respectively designed in according to PoIFN total length which was amplified by our lab and NheⅠand HindⅢwere designed in the primmers.PoIFN-α/β/γsegments of sequences was amplified by PCR,three segments of sequences were subloned to pcDNA3.1-IFN vector,and those linked vectors were transformed into E.coli DH5α.The positive clones were verified from PCR with bacterium,the conclusions of sequence determined showed that poIFNs were cloned. Bacterium was amplified culture then extract plasmid and purfied the plasmids.2.Three pairs primmer were respectively designed in according to PoIFN total length which was amplified by our lab and EcoRⅠand HindⅢwere designed in the primmers.PoIFN-α/β/γsegments of sequences was amplified by PCR,three segments of sequences were subloned to AAV vetor,and those linked vectors were transformed into E.coli.DH5α.The positive clones were verified from PCR with bacterium,Amplifying culture then extract plasmid and purfied the plasmids.3.Transfection of PoIFN in 293T cellThree plasmids system（AAV-PoIFN,AAV-RC,AAV-pHelper,mixed with 1:1:1） and pcDNA3.1-PoIFN plasmids were transfected in 293T cell wirh lipofectamine^TM2000.The IFN protein was detected by western-blot.更多还原