猪γ-干扰素在重组杆状病毒中的表达及其抗病毒活性的测定  

Expression of Porcine Gamma-interferon in Recombinant Baculovirus and Determination of Its Antiviral Activity

作　　者：秦立廷[1,2] 王喜军[1] 胡森[1] 李志中[1,3] 陈伟业[1,4] 葛金英[1] 刘思当[5] 步志高[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室 [2]山东农业大学动物科技学院,泰安271018 [3]内蒙古农业大学动物科学与医学学院,呼和浩特010018 [4]南京农业大学动物医学院,南京210095 [5]山东农业大学动物科技学院

出　　处：《生物工程学报》2007年第3期386-391,共6页Chinese Journal of Biotechnology

基　　金：国家"攻关"项目(No.2004BA519A19;2005BA711A10);国家"973项目"(No.2005CB523200)资助。~~

摘　　要：研究利用Bac-To-Bac杆状病毒表达系统构建含有猪γ-干扰素(porcine interferon-γ,PoIFN-γ)完整开放阅读框的供体质粒pFastBacTM1-PoIFN-γ,转化DH10Bac感受态细胞获得重组穿梭质粒rBacmid-PoIFN-γ,转染sf9昆虫细胞救获表达PoIFN-γ的重组杆状病毒rBac-PoIFN-γ。以抗PoIFN-γ单克隆抗体为一抗进行Western blot、间接免疫荧光(IFA)及间接ELISA检测,结果表明PoIFN-γ在重组杆状病毒rBac-PoIFN-γ感染的昆虫细胞中获得正确表达。抗病毒活性试验显示,重组杆状病毒表达rPoIFN-γ能有效抑制水疱性口炎病毒(VSV)在猪肾细胞系(PK-15)的复制,rBac-PoIFN-γ感染昆虫细胞培养上清抗病毒活性为2×104抑制单位(IU)/mL,其抗病毒活性可以被鼠抗原核表达重组PoIFN-γ免疫血清有效阻断。结果表明rPoIFN-γ在重组杆状病毒rBac-PoIFN-γ在感染昆虫细胞获得有效表达,并具有高效抗病毒活性。The full-length porcine interferon gamma（PoIFN-γ） cDNA, including the secretion signal peptide coding region was recloned into honor plasmid pFastBac^TM 1 of Bac-To-Bac Baculovirus Expression System. These recombinant plasmids, pFastBacTM 1-PoIFN-γ, were transformed into DH10Bac host bacteria to get recombinant shuttle plasmids, rBacmid-PoIFN-γ. Recombinant baculovirus, rBac-PoIFN-γ, was generated for expressing PoIFN-γ, by transfecting rBacmid-PoIFN-γ with Cellfectin Reagent into sf9 insect cells. The expression of PoIFN-γ in insect cells was confirmed by Western Blot, indirect immunofluorescence assay and indirect ELISA. The antiviral activity assay shows that PoIFN-γ expressed by the rBac-PoIFN-γ can efficiently inhibit the replication of the recombinant Vesicular Stomatitis Virus expressing green fluorescence protein in PK-15 cells. The antiviral activity of PoIFN-γ can be specifically blocked by anti-PoIFN-γ mouse serum. The antiviral titer of culture supematant of insect cells infected by rBac-PoIFN-7 is 2 × 10^4 IU/mL. The results demonstrat that the rBac-PoIFN-γ can express rPoIFN-γ efficiently and rPoIFN-γ has high antiviral activity.

关 键 词：Γ-干扰素 猪 重组杆状病毒 抗病毒活性 

分 类 号：Q789[生物学—分子生物学] S852.5[农业科学—基础兽医学]