牛γ-干扰素在大肠杆菌和毕赤酵母中的表达和抗病毒活性比较.pdfVIP

业生物技术学报 Journal of Agricultural Biotechnology 2005,13 (4): 46 1~467 ·研究论文· 牛 - 干扰素在大肠杆菌与毕赤酵母中的表达及抗病毒活性比较* 喜菊 张 灿 韩春来 夏 春 汪 明** （中国农业大学动物医学院，北京 100094 ） ： 摘要 用RT-PCR 从经ConA 刺 的荷斯坦奶牛外周血淋巴细胞总RNA 中扩增了 基因，克隆入pGEM T-easy 载体 测序。再亚克隆其成熟肽基因，分别构建大肠杆菌( )表达载体pET28a/ 和毕赤酵母( )表达载体 pPICZ琢/ 。前者在 BL21 中经IPTG 诱导实现了高效表达，表达蛋白占菌体总蛋白的30%，表达产物以包涵体形式存； 后者在 GS115 中经甲醇诱导实现了高效分泌表达，表达产物直接分泌到培养上清中，表达量约为1.0 g/L。分别在 CEF/VSV 和MDBK/VSV 细胞系上对2 种表达蛋白的抗病毒活性进行了比较，结果表明, 两种重组蛋白在MDBK/VSV 抗病毒活 性高于在CEF/VSV 上的活性，而毕赤酵母表达的蛋白抗病毒活性高于大肠杆表达蛋白，约为前者的2 倍。 ： ；毕赤酵母；抗病毒活性 关键词 牛 - 干扰素；分子克隆；大肠杆菌 Expressions of Bovine Interferon- in and and Comparision of Their Antiviral Activities SHI Xi-Ju ZHANG Can HAN Chun-Lai XIA Chun WANG Ming** Bovine interferon- gene was cloned from the total RNA of peripheral blood lymphocytes stimulated with ConA using RT-PCR and cloned into pGEM T-easy vector and sequenced. The mature peptide without signal was then subcloned and constructed into the expression plasmids of pET28a/ and pPICZ / , respectively, and the former was highly expressed with 30% of bacterial whole proteins in inclusion body after induced by IPTG in BL2 1; The latter was expressed in GS115 with methanol induction and the expressed products were directly secreted into cultural supernatant with the expression level of 1.0 g/L. Antiviral activities analyzed in CEF/VSV and MDBK/VSV