悬浮培养Sf21细胞高效表达牛γ-干扰素及其特性鉴定

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pFastBacTMHTA中构建重组质粒pFastBacTM HTA-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rBovIFN-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 recom-binant plasmid, named as pFastBacTM HTA-lFN-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 rBovIFNγ 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.