奶牛γ-干扰素基因克隆及其在大肠杆菌中表达  

CLONING AND EXPRESSION OF BOVINE INTERFERON-GAMMA GENE IN ESCHERICHIA COLI

作　　者：许金俊[1] 秦爱建[1] 刘岳龙[1] 金文杰[1] 李瑞芳[1] 

机构地区：[1]扬州大学畜牧兽医学院动物医学系,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2003年第1期5-9,共5页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：江苏省"3 3 3"工程人才培养基金资助项目 ( 2 0 0 2 2 2 )

摘　　要：根据奶牛γ干扰素 ( Bov IFN-γ)基因的 c DNA序列设计 1对引物 ,应用一步法逆转录聚合酶链式反应 ( RT-PCR)技术从奶牛脾淋巴细胞的总 RNA中扩增出特异性片段。将 RT-PCR产物克隆到 Pucm-T载体中 ,经过限制性酶切分析、测序证实扩增得到的 Bov IFN-γ基因序列正确。将该基因片段切下并克隆到大肠杆菌表达载体 PGEX-6P-1谷光甘肽 S 转移酶基因的下游 ,经 IPTG诱导后 ,表达出 42 ku的融合蛋白 ,薄层扫描测定可溶性融合蛋白表达量约占菌体可溶性总蛋白的 2 5 %。通过细胞病变抑制试验证实 ,融合蛋白具有较好的生物学活性 ,在牛肾细胞上抑制水泡性口炎病毒的活性可达到 1 63 84U· mg- 1 ,有望成为预防和控制奶牛疾病的新产品。Bovine interferon gamma (BovIFN γ ) gene without signal peptide encoding region was amplified by reverse transcription polymerase chain reaction (RT PCR) from total RNA of bovine spleen lymphocytes stimulated with Phytohemagglutinin (PHA). The products of RT PCR were cloned into Pucm T vector. Postive recombinant clones named BovIFN γ T(1 3) were identified by restriction enzyme digestion and sequencing. The fragment of BovIFN γ in the Pucm T vector was subcloned into PGEX 6P 1. Partly soluble 42 ku GST BovIFN γ fusion protein was expressed after induced by IPTG, which was confirmed by SDS PAGE and accounted for 25% of total soluble bacterial proteins by Tlc scanning. Recombinant fusion protein showed some antiviral activity in the vesicular stomatitis cytopathic effect reduction assay. The interferon titer in the Supernatant of bacterial sonicates was up to 16 384 U·mg -1 .

关 键 词：奶牛 基因克隆 大肠杆菌 基因表达 Γ-干扰素基因 

分 类 号：S823.91[农业科学—畜牧学] Q785[农业科学—畜牧兽医]