奶牛γ-干扰素基因的克隆及其在COS-1细胞中的表达  

Cloning of Bovine Interferon-gamma Gene and Its Expression in COS-1 Cells

作　　者：许金俊[1] 秦爱建[1] 金文杰[1] 刘岳龙[1] 

机构地区：[1]扬州大学畜牧兽医学院,扬州225009

出　　处：《农业生物技术学报》2004年第3期273-277,共5页Journal of Agricultural Biotechnology

基　　金：江苏省"333"工程人才培养基金资助

摘　　要：应用RT-PCR技术从经体外植物血凝素(PHA)刺激诱导的奶牛外周血淋巴细胞的总RNA中扩增出牛γ-干扰素(bovineinterferon-γ,BovIFN-γ)基因。将扩增出的BovIFN-γ克隆到PMD18-T载体中,经过限制性酶切分析筛选正向插入的克隆。测序结果表明,所克隆到的基因编码区序列与已报道基因存在3个核苷酸的差异。将该基因片段从PMD18-T载体中用HindⅢ和BamHⅠ双酶切后克隆到真核表达载体pcDNA3.1/zeoc(+)中,构建真核表达载体pcDNA-BovIFN-γ,通过脂质体转染COS-1细胞,用鼠抗BovIFN-γ高免血清和FITC标记的羊抗鼠IgG进行间接免疫荧光实验(IFA)检测,结果表明,在COS-1细胞的细胞膜和胞浆内均有rBovIFN-γ表达,细胞病变抑制实验证实,转染后细胞培养上清液具有一定的干扰素活性,转染后72h细胞培养上清液在MDBK细胞上抑制VSV病毒的效价可达到128IU/mL。研究结果为进一步筛选稳定表达BovIFN-γ基因细胞系、开发奶牛疾病预防控制的新产品以及开展奶牛疾病的基因治疗奠定了基础。Bovine interferon-gamma(BovIFN-γ) gene with signal peptide encoding region was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of bovine peripheral blood lymphocytes stimulated with phytohemagglutinin (PHA). The products of RT-PCR were cloned into PMD18-T vector. Postive recombinant clone named BovIFN-γ-T4 was identified by restriction enzyme digestion and sequencing. The fragment of BovIFN-γin the PMD18-T vector was subcloned into mammalian expression vector pcDNA3.1, which was named with pcDNA-BovIFN-γ. Eukaryotic expression plasmid pcDNA-BovIFN-γwere transfected into COS-1 cells by lipofectin, rBovIFN-γexpression was identified in the cytoplasms and cytomembranes of COS-1 cells in IFA at 72 h posttransfection. The interferon titer in the supernatant of transfected COS-1 cells was up to 128 IU/mL. These results suggested that pcDNA-BovIFN-γcould be used to establish a cell line expressing rBovIFN-γstably, and rBovIFN-γ could be served as a new product in the control of bovine diseases.

关 键 词：奶牛 Γ-干扰素 基因 克隆 COS-1细胞 表达 植物血凝素 

分 类 号：Q78[生物学—分子生物学]