蜂素信号肽介导猪干扰素-γ在杆状病毒中分泌表达及抗病毒活性检测  

Secretory expression of porcine interferon-γ in a baculovirus using HBM signal peptide and detection of its antiviral activity

作　　者：王彦彬[1] 魏战勇[1] 陈红英[1] 王建举[1] 郭小参[1] 崔保安[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《中国兽医学报》2010年第11期1440-1445,共6页Chinese Journal of Veterinary Science

基　　金：国家"十一五"科技支撑计划资助项目(2006BAD06-A08)

摘　　要：应用Bac-to-Bac杆状病毒/昆虫细胞表达系统,将编码成熟的猪干扰素-γ基因插入供体质粒pFastBacTM1polyhedrin启动子控制下的多克隆位点,引入昆虫细胞可识别的蜂素信号肽(Honeybee melittin signal peptide)取代猪干扰素-γ原有信号肽以实现在昆虫细胞中分泌型表达,并在C端融合6个组氨酸标签以利于纯化。将构建质粒转化DH10Bac感受态细胞获得重组穿梭载体质粒,转染对数生长期的Sf9昆虫细胞获得重组杆状病毒。重组蛋白通过SDS-PAGE、间接免疫荧光、Western-blot证明在重组杆状病毒感染的昆虫细胞中获得分泌表达。通过在猪肾细胞(PK-15)上抑制水泡性口炎病毒(VSV)致病变作用检测重组蛋白的抗病毒活性是1.38×106U/mL。The gene sequences encoding mature porcine interferon-gamma（PoIFN-γ） fused with a C-terminal 6×Histidine tag were cloned into the baculovirus pFastBacTM1 vector of the Bac-to-Bac baculovirus expression system under the control of pH promoter,The authentic signal sequences of porcine interferon-gamma were substituted with the honeybee melittin signal sequence,and expressed in insect cells.The recombinant proteins were detected in expressing cells by immunofluorescence assay and Western-blot analysis.The rPoIFN-γ purified by nickel affinity spin column was a 19 000 protein as confirmed by SDS-PAGE.Bioactivity of the recombinant PoIFN-γ were verified to be cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in PK-15 cells,which is about 1.38×106 U/mL.The dilution of 27 of rPoIFN-γ in supernant and insect cells can protect the Marc-145 from the cytopathic effect cause by 100TCID50 of porcine reproductive and respiratory syndrome virus.The recombinant proteins will be explored as a poteintial therapeutic agent and immunological adjuvant.

关 键 词：猪 干扰素 杆状病毒 抗病毒活性 

分 类 号：Q78[生物学—分子生物学] S852.65[农业科学—基础兽医学]