悬浮培养Sf21细胞高效表达牛γ-干扰素及其特性鉴定  

Efficient Expression and Characterization of Bovine Interferon-γ in Suspension Sf21 Cells

作　　者：王海春[1] 贾红[1] 袁维峰[1] 侯绍华[1] 郭晓宇[1] 鑫婷[1] 朱鸿飞[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国畜牧兽医》2012年第11期1-6,共6页China Animal Husbandry & Veterinary Medicine

基　　金：公益性行业(农业)科研专项经费项目(200903027);863计划(SQ2011AAJY2746;2012AA101302);中央级公益性科研院所基本科研业务费(2010js-1;2011js-3);"十一五"国家科技支撑计划重点项目(2010BAD04B02);2011年及2012年农业部动物疫情监测与防治项目

摘　　要：本试验旨在研究重组牛γ-干扰素(rBovIFN-γ)在Sf21细胞中高效表达及其特性鉴定。利用逆转录—聚合酶链式反应(RT-PCR)技术从奶牛外周血淋巴细胞的总RNA中扩增出不含信号肽的BovIFN-γ基因片段。将其克隆到pFast-BacTMHTA中构建重组质粒pFastBacTMHTA-BovIFN-γ。然后将重组质粒转化至DH10Bac感受态细胞获得重组穿梭质粒rBacmid-BovIFN-γ。转染Sf21昆虫细胞后获得重组杆状病毒rBac-BovIFN-γ,应用Sf21细胞悬浮培养技术大量表达rBov-IFN-γ。利用Western blotting、质谱分析(MS)、抗病毒试验等对rBovIFN-γ进行鉴定。Western blotting分析显示,rBovIFN-γ具有良好的反应原性;MS分析表明,rBovIFN-γ的6个肽段序列与牛IFN-γ的氨基酸序列完全匹配,在蛋白质质谱数据库中搜索结果为牛IFN-γ;MDBK/VSV抗病毒试验显示,rBovIFN-γ具有很高的抗病毒活性,为1.05×105 IU/mg。In the study,we mainly did the research about efficient expression and characterization of bovine interferon-γ in suspension Sf21 cells.Bovine IFN-γ gene was amplified by RT-PCR,and inserted into the pFastBacTM HTA to get the recombinant plasmid,named as pFastBacTMHTA-IFN-γ.After transformation and transfection,the recombinant baculovirus was obtained.The target protein named rBovIFN-γ was successfully expressed in Sf21 insect cells during suspension culture.We identified the rBovIFN-γ with SDS-PAGE,Western blotting and mass spectrum（MS）.Western blotting analysis showed that the recombinant protein had good activity.The protein MS identification results confirmed the recombinant bovine IFN-γ expression via the baculovirus expression system.MDBK/VSV antiviral test results showed that the rBovIFN-γ had high antiviral activity at 1.05×105 IU/mg.

关 键 词：牛γ-干扰素 杆状病毒 悬浮培养 质谱分析 抗病毒活性 

分 类 号：Q78[生物学—分子生物学]