鸡γ干扰素基因的克隆和表达及其多克隆抗体的制备-预防兽医学专业论文.docxVIP

摘 摘 要 摘 要 Y．干扰素(Interferon，y、IFN—y)主要由滢忱的T鲴腿及NK细腿产生，具礴抗病毒作 带，也其有上调MHC Il(major histoeompatibility eomptex，MHC)类抗琢分子的表达等作麓， 是影响机体细胞免疫和体液免疫反应的一类具有多种调节效应的细胞因子。 本试验根据鬻辨已发袭豹序列，设计一对；|物，琏耀逆转聚．聚合酶链式爱巍(Revels} traascrlption-potymerase chain reaction，RT-PCR)技术，从刀豆素A(Coneanavatin A，Con A、 诱导培莽24h的鹏脾淋巴细胞中扩增得到了大小约500bp的基因，将其连接到pUCl9质粒上， 经震粒PCR手珏EcoR l，Hind HI霰酶甥鉴定螽，簿选粥性重组毙瘫。序列分辑表鹤，该基蕊 阅读开放框架由492个核营酸组成，与马和人IFN．y撼因序列的同源性分别为35％和32％， 与鸡I型干扰紊纂因的厨源性仅为25％，与隔，}报道的鸡IFN．y基因序列完全一致。这表明 鸡簿海融细脆受委n有丝分裂原刺激螽，其IFN-y基因mRNA获褥了表达。本试验成功克隆榴 到了鸡IFN．y基因。 憋竟隧褥到的鸡IFN—y基霞亚毙隆戮丈强抒莲擐援表达载热pPROEX“}拜粒Pst l霜 Kpnl位点上，构建重组表达质粒pPROEX HT-IFN-Y。经序列分析鉴斑，确证目的基因克 隆到载体的预期位点。将重组质粒转化大肠杆菌DH5 a，于37C诱导培莽8h，SDS．PAGE凝 胺电滚衰鹈该基戮在大瑟稃蘩孛获褥了蹇水警表达，表达静鸡IFN．￥融含蟹彦分予踅约为22 ku。该重组蛋白占菌体总蛋白的33％。经Western blot浆定，确定该重组骚白为包涵体，具有 生物学潘性。该包涵体被6M盐酸胍裂解后，通过镍离予亲和树脂进行了纯化。用掰获得豹重 缮鸡IFN*￥融合骚自及其纯纯产物缀三次飘肉注射新西兰白兔。错《备兔抗鸿IFN-Y多克隆抗 体。琼脂扩散实验结果表明，多克隆抗体可岛纯化的鸩IFN．Y菔组蛋自发生特异性反应。 本次试验残功宠隆、表达、纯毒{：了鸡IFN-￥鞋台甏宣，著；蠡l}鍪了兔撬避IFN-￥多竞隆撬 皿清，可望用于端IFN—Y薰组蛋自艇物学特性及其应用的研究中。)。。 关键壤鸿；烈”y：基戮究隆；固重缀簧鑫； 彳 ．V* Cloning Cloning and Expression of the Chicken Y—interferon Gene and the Preparation of Polyclonal Antibody against Recombinant Chicken IFN—Y Abstract Interferon．y is an anfiviral cytokine produced by activated T eell and NK cell．1t upregulated MHC珏antigen expression，influenced the cellular and humoral immune response and has pleiotropic regulatory effects on immune system． One gene was amplified by reverse transcription—polymerase chain reaction(RT-PCRl in splenocytes which was stimulated with CorLA．for 24 hours．The gene was cloned into PUel9 vector and identified with PCR and Eco RI+Hind III digestion．Then．one of the POsitive recombinant clone was sequenced and analysed．The result showed that i扭sequence was 35％ and 32％ldentical to equine IFN-￥and human lFN—y respectively,but it only shared l 5％ homology with chicken type I IFN．The sequence was jast the same as the published chicken IFN—Y gene．So the chicken IFN一￥mRNA may be expressed in ConA-activated chicken splenocytes and the IF