鸡源IFIT5蛋白的体外表达及单克隆抗体的制备  

In vitro expression of chicken interferon-induced protein with tetratricopeptide repeats 5(IFIT5) and preparation of its monoclonal antibody

作　　者：高向向[1] 贾璐[1] 胡伯里[1] 邓婷娟 郑肖娟[1] 周继勇[1] 

机构地区：[1]浙江大学农业部动物病毒学重点实验室,浙江杭州310058

出　　处：《中国兽医科学》2016年第9期1087-1093,共7页Chinese Veterinary Science

基　　金：国家蛋鸡产业技术体系岗位专家项目(CARS-41-K11)

摘　　要：鸡IFIT5(chIFIT5)蛋白在抗病毒免疫中具有重要作用。为了阐明其具体作用机制,本研究将禽传染性法氏囊病病毒(infectious bursal disease virus,IBDV)感染DF-1细胞后,通过RT-PCR方法扩增chIFIT5基因,将其构建到pET-28a(+)原核表达载体上进行表达,然后用纯化的重组chIFIT5蛋白免疫小鼠后通过细胞融合技术筛选能稳定分泌抗chIFIT5蛋白的单克隆抗体细胞株。结果显示,扩增获得的chIFIT5基因的大小为1 440 bp,在原核细胞中成功表达的重组chIFIT5蛋白约58 ku。Western-blot检测显示,有4株单抗与重组chIFIT5蛋白、IBDV感染的细胞中chIFIT5蛋白以及Myc-chIFIT5真核转染蛋白均能发生免疫反应,但间接免疫荧光(IFA)检测反应却均呈阴性。抗体亚类分析结果显示,四株细胞株所分泌的单克隆抗体均为Ig G 2a亚类,轻链均为κ链。这为鸡源IFIT蛋白的生物学特性研究奠定了基础。The chicken interferon-induced protein with tetratricopeptide repeats 5（chIFIT5） plays vital roles in mediating antiviral immune response. To elucidate its antiviral mechanisms,the chIFIT5 gene was amplified by RT-PCR from IBDV-infected DF-1 cells and cloned into prokaryotic expression vector p ET-28a（＋） to express recombinant protein. After immunizing BALB/ c mice with the purified recombinant chIFIT5,we screened monoclonal hybridoma cell st rains whichcould secret ant ibodies against chIFIT 5.Result s showed t he lengt h of amplifided chIFIT5 gene was 1 440 bp and through prokaryotic expression, the recombinant chIFIT5 protein with a molecular weight of 58 ku was obtained. There were four hybridoma cell strains whichcould react with the recombinat chIFIT5 protein, chIFIT5 in IBDV-infected DF-1cells and Myc-IFIT5-transfected 293 T cells by Western-blot,but no reaction was detected in immunofluorescence assay. Al four hybridoma cel strains against chIFIT5 belonged to Ig G2 a subtype with κ chain. The m Abs might provide useful tools for further biological function studies on chIFIT5.

关 键 词：IFIT家族蛋白 chIFIT5 单克隆抗体 抗体亚型 

分 类 号：S852.43[农业科学—基础兽医学]