【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。更多还原

【Abstract】 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.更多还原