猪γ-干扰素的克隆表达及单抗的潜在应用  

Preparation and Potential Application of Monoclonal Antibody Against Porcine IFN-γ Cloned and Expressed in E. coli

作　　者：娄忠子[1] 兰喜[1] 李建强[1] 李学瑞[1] 李志勇[1] 殷相平[1] 柳纪省[1] 

机构地区：[1]中国农业科学院兰州兽医研究所传染病室家畜疫病病原生物学国家重点实验室农业部草食动物疫病重点开放实验室农业部兽医公共卫生重点开放实验室,兰州730046

出　　处：《生物技术通报》2010年第1期173-179,共7页Biotechnology Bulletin

基　　金：科研院所社会公益研究专项(2005DIB4J041)

摘　　要：以刀豆素A(ConA)刺激长白猪外周血单个核细胞(PBMC),提取的总RNA为模板,采用一步法RT-PCR扩增出包含猪γ-干扰素(PoIFN-γ)完整基因的片段。将完整的PoIFN-γ基因亚克隆到pET-32a(+)上,构建pET-IFN-γ融合表达载体,转化入Rosetta(DE3)后IPTG诱导表达,得到大小约为36.8kD的目的蛋白。将目的蛋白检测及纯化后免疫后BALB/c小鼠,取脾细胞与SP2/0细胞融合,最终筛选出3株能稳定分泌抗猪IFN-γ单克隆抗体的杂交瘤细胞。ELISA叠加试验显示,3株单抗针对相同或邻近的PoIFN-γ抗原表位,制备的腹水中单抗间接ELISA效价为1×104。选取单抗IF-2株进行单抗的潜在应用研究,Westernblotting分析结果显示单抗IF-2株能与融合表达蛋白产生特异性反应,与pET-32a(+)标签蛋白无反应;间接免疫荧光检测(IFA)发现,在采用腺病毒表达系统表达PoIFN-γ的HEK-293细胞中散在大量特异性荧光。The total RNA was extracted as template from peripheral blood mononuclear cells（PBMC）which were isolated from Landrace pigs and stimulated with concanavaline A （ConA） to amplify porcine interferon gamma gene （PolFN-γ）. The amplicon which contained the complete PoIFN-γ gene was amplified by one step RT-PCR. The complete PoIFN-γ, gene was subcloned into pET-32a （ ＋ ）to construct pET-IFN-γ recombinant fusion expression vector. The recombinant vector was transformed into Rosetta（ DE3 ）E. coli and expressed by induction of IPTG. The molecular weight of recombinant protein were 36. 8 kD detected by SDS-PAGE. After detection and purification, the fusion protein was utilized to immunize BALB/c mice, and spleen cells were collected to fuse with SP2/0 myeloma cells. Three monoclonal hybridoma cell lines that stably secreted monoclonal antibodies were generated after a series of screening. ELISA additivity test indicated that the three strains of monoclonal antibodies recognized a same or nearby epitope. The three ascitic - mAbs have a same antibody titre of 1 104 detected by indirect ELISA. IF-2 strain was selected to develop potential application by westera blotting and IFA. Positive signal could be detected between expressed PoIFN-γ, and IF-2, but negative signal between His-tag protein and IF-2 in western blotting;specific fluorescence could be detected in HEK-293 cells inoculated with recombinant adenovirus which were constructed for expressing PoIFN-γ in IFA.

关 键 词：猪Γ-干扰素 融合表达 单克隆抗体 潜在应用 

分 类 号：Q789[生物学—分子生物学] S852.5[农业科学—基础兽医学]