施马伦贝格病毒核衣壳蛋白在昆虫杆状病毒表达系统中的表达与纯化  

Expression and purification of Schmallenberg virus nucleocapsid protein using the Bac-to-Bac baculovirus expression system

作　　者：宋姗姗[1] 张永宁[1] 吴绍强[1] 林祥梅[1] 

机构地区：[1]中国检验检疫科学研究院动物检疫研究所,北京100029

出　　处：《中国兽医科学》2016年第4期442-448,共7页Chinese Veterinary Science

基　　金："十二五"国家科技支撑计划项目(2013BAD12B01);中国检验检疫科学研究院"青年英才计划"项目(CAIQ-YC-20140205);国家质检总局科技计划项目(2013IK054)

摘　　要：为表达与纯化具有天然构象的施马伦贝格病毒(SBV)核衣壳(N)蛋白,在SBV-N蛋白编码基因的N端引入谷胱甘肽-S-转移酶(GST)标签编码序列,将其克隆至杆状病毒表达载体pFastBac 1中构建重组供体质粒pFastBac-GST-SBV-N;经测序鉴定后,将pFastBac-GST-SBV-N转化大肠杆菌DH10Bac感受态细胞,使其与DH10Bac内含有的杆状病毒穿梭载体(杆粒)进行位点特异性转座,形成重组杆粒Bacmid-GST-SBV-N;抽提重组杆粒,转染Sf9昆虫细胞制备表达GST-SBV-N融合蛋白的重组杆状病毒rBac-GST-SBV-N;利用间接免疫荧光试验检测rBac-GST-SBV-N感染的Sf9细胞与SBV抗血清的反应情况。结果表明,rBac-GST-SBV-N感染的Sf9细胞能够与SBV抗血清发生特异性反应。利用谷胱甘肽琼脂糖凝胶4B纯化Sf9细胞内表达的GST-SBV-N融合蛋白,借助Pre Scission蛋白酶切除GST标签后,对重组SBV-N蛋白进行Western-blot鉴定和间接ELISA分析。结果显示,在分子质量约为26 ku处出现一条特异性条带,其大小与SBV-N蛋白相符;该蛋白与抗SBV-N蛋白单克隆抗体2C8和SBV抗血清均能发生特异性反应,说明该蛋白具有良好的反应原性。SBV-N真核表达蛋白的成功制备和纯化,为施马伦贝格病血清学检测方法的建立提供了诊断抗原。To express and purify Schmallenberg virus（SBV） nucleocapsid（N） protein with native conformation,the SBV-N gene with an inserted coding sequence for an N-terminal glutathione-S-transferase（GST） tag was cloned into the pFastBac 1 vector to construct recombinant donor plasmid pFastBac-GST-SBV-N.After sequence confirmation,the donor plasmid was transformed into Escherichia coli DH10 Bac competent cells,in which site-specific transposition occurred between the mini-Tn7 element on the pFastBac 1 vector and the mini-att Tn7 target site on the baculovirus shuttle vector（Bacmid）.This ultimately formed a recombinant Bacmid-GST-SBV-N,which was then transfected into Sf9 insect cells to package recombinant baculoviruses that express the GST-tagged SBV-N fusion protein（designated rBac-GST-S BV-N）.Indirect immunofluorescence assays revealed that the rBac-GST-SBV-N-infected Sf9 cells reacted specifically with SBV antisera.The GST-SBV-N fusion protein expressed in Sf9 cells was purified using Glutathione Sepharose 4B and the GST tag was successfully removed by cleavage with the Pre Scission Protease.The purified SBV-N protein was then analyzed by Western-blot analyses and indirect enzyme-linked immunosorbent assays.The results demonstrated that a specific protein band appeared at the relative molecular weight of around 26 ku,which corresponded to a theoretical molecular weight for SBV-N protein,and that the target protein reacted specifically with both anti-SBV-N monoclonal antibody 2C8 and SBV antisera.These results indicate that the N protein has good reactionogenicity.The successful preparation of eukaryotic SBV-N protein provides a valuable biological material that has potential for the serological diagnosis of Schmallenberg disease.

关 键 词：施马伦贝格病毒 核衣壳蛋白 杆状病毒表达载体 昆虫细胞 蛋白纯化 

分 类 号：S852.659.5[农业科学—基础兽医学]