【作者】 丁忠庆；

【导师】 宋铭忻；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 γ-干扰素（Interferon-γ，IFN-γ）主要由活化的T细胞及NK细胞产生，具有抗病毒作用，也具有上调MHCⅡ（major histocompatibility complex, MHC）类抗原分子的表达等作用，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。 本试验根据国外已发表的序列，设计一对引物，应用逆转录-聚合酶链式反应（Reverse transcription-polymerase chain reaction，RT-PCR）技术，从刀豆素A（Concanavalin A，Con A）诱导培养24h的鸡脾淋巴细胞中扩增得到了大小约500bp的基因，将其连接到pUC19质粒上，经质粒PCR和EcoR Ⅰ、Hind Ⅲ双酶切鉴定后，筛选阳性重组克隆。序列分析表明，该基因阅读开放框架由492个核苷酸组成，与马和人IFN-γ基因序列的同源性分别为35％和32％，与鸡I型干扰素基因的同源性仅为15％，与国外报道的鸡IFN-γ基因序列完全一致。这表明鸡脾淋巴细胞受到有丝分裂原刺激后，其IFN-γ基因mRNA获得了表达。本试验成功克隆得到了鸡IFN-γ基因。 将克隆得到的鸡IFN-γ基因亚克隆到大肠杆菌原核表达载体pPROEX^TMHT的Pst I和KpnI位点上，构建重组表达质粒pPROEX^TMHT-IFN-γ。经序列分析鉴定，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DH5α，于37℃诱导培养8h，SDS-PAGE凝胶电泳表明该基因在大肠杆菌中获得了高水平表达，表达的鸡IFN-γ融合蛋白分子量约为22ku。该重组蛋白占菌体总蛋白的33％。经Western blot鉴定，确定该重组蛋白为包涵体，具有生物学活性。该包涵体被6M盐酸胍裂解后，通过镍离子亲和树脂进行了纯化。用所获得的重组鸡IFN-γ融合蛋白及其纯化产物经三次肌肉注射新西兰白兔，制备兔抗鸡IFN-γ多克隆抗体。琼脂扩散实验结果表明，多克隆抗体可与纯化的鸡IFN-γ重组蛋白发生特异性反应。 本次试验成功克隆、表达、纯化了鸡IFN-γ融合蛋白，并制备了兔抗鸡IFN-γ多克隆抗血清，可望用于鸡IFN-γ重组蛋白生物学特性及其应用的研究中。更多还原

【Abstract】 Interferon- is an antiviral cytokine produced by activated T cell and NK cell. It upregulated MHC II 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-PCR) in splenocytes which was stimulated with ConA for 24 hours. The gene was cloned into pUC19 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 its sequence was 35% and 32% identical to equine IFN- and human IFN- respectively, but it only shared 15% homology with chicken type I IFN. The sequence was just the same as the published chicken IFN- gene. So the chicken IFN- mRNA may be expressed in ConA-activated chicken splenocytes and the IFN- Y gene had been cloned from chicken splenocytes successfully.The cDNA of chicken IFN- Y gene was subcloned into Pst I and Kpn I sites of pPROEX橦T vector to construct recombinant plasmid pPROEX橦T-IFN- . The recombinant plasmid was translated into E. coli DH5 and the bacteria was induced with IPTG. It was demonstrated by SDS-PAGE and Western blot that a 22ku protein which was equal to chicken IFN- y protein in molecular weight was expressed in E.coli DH5 . This protein was purified and used to prepare the antibody against chicken IFN- y protein. The result showed that the antibody could react with purified recombinant chicken IFN- protein.更多还原