牛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 bovine interferon gamma in recombinant baculovirus and determination of its antiviral activity

摘要:

研究利用Bac-To-Bac杆状病毒表达系统构建含有牛γ-干扰素(Bovine interferon-γ,BoIFN-γ)完整开放阅读框的供体质粒pFastBacTM1-BoIFN-γ,转化DH_10Bac感受态细胞获得重组穿梭质粒rBacmid-BoIFN-γ,转染sf9昆虫细胞救获表达BoIFN-γ的重组杆状病毒rBac-BoIFN-γ。采用抗BoIFN-γ单克隆抗体作为一抗进行间接免疫荧光(IFA)及间接ELISA检测,表明BoIFN-γ在重组杆状病毒rBac-BoIFN-γ感染的sf9昆虫细胞中获得正确表达。利用VSV*GFP-MDBK细胞系统测定rBoIFN-γ抗病毒活性,重组杆状病毒表达重组BoIFN-γ(rBoIFN-γ)能有效抑制水疱性口炎病毒(VSV)在牛肾细胞(MDBK)上的复制,rBac-BoIFN-γ感染sf9昆虫细胞上清抗病毒活性为2×105IU/mL,而且其抗病毒活性可以被鼠抗原核表达重组BoIFN-γ免疫血清阻断。结果表明:rBoIFN-γ在重组杆状病毒rBac-BoIFN-γ感染的sf9昆虫细胞中获得良好表达,并具有高效抗病毒活性。

Abstract:

The full-length bovine interferon gamma(BoIFN-γ) cDNA，including the secretion signal peptide coding region was recloned into baculovirus honor vectors pFastBac^TM1 of Bac-To-Bac system. These recombinant plasmids，pFastBac^TM1-BoIFN-γ，were transformed into DH10Bac host bacteria to get recombinant shuttle plasmids，rBacmid-BoIFN-γ. Recombinant baculovirus，rBac-BoIFN-γ，was generated for expressing BoIFN-γ，by transfecting recombinant Bacmid-BoIFN-γ with CellfectinRReagen into sf9 insect cells. BoIFN-γ efficiently expressed by recombinant baculovirus in sf9 cells was testified by indirect immunofluorescence assay and indirect ELISA with monoclonal antibody against Bovine interferon-γ. Furthermore，VSV*GFP，recombinant Vesicular Stomatitis Virus expressing green fluorescence protein and MDBK were used to determine the anti-viral activity of rBoIFN-γ. The result shows rBoIFN-γ could inhibit the replication of the VSV*GFP in MDBK cells and the antiviral activity of supernatant was 2×10⁵IU/mL. The antiviral activity of rBoIFN-γ could be blocked by anti-BoIFN-γ mouse serum. The results demonstrated that the recombinant baculovirus could express BoIFN-γ efficiently and rBoIFN-γ had high antiviral activity.