猪干扰素α在昆虫细胞中分泌表达及其抗病毒活性检测

 【目的】为获得重组猪干扰素α。【方法】本研究应用Bac-to-Bac杆状病毒/昆虫细胞表达系统,将编码成熟猪干扰素α基因插入供体质粒pFastBacⅠ多克隆位点，置于pH启动子控制下，昆虫可识别的蜂素信号肽（honeybee melittin signal peptide，HBM）取代猪干扰素α原有信号肽以实现分泌型表达，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DH10感受态细胞进行同源重组，经抗性和蓝白斑筛选，获得重组穿梭质粒Bacmid，转染对数生长期的Sf9昆虫细胞获得重组杆状病毒。【结果】重组蛋白通过间接免疫荧光、Western-blot证明重组蛋白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，结果表明：昆虫细胞上清的抗病毒效价达到1.07×105 U?ml-1，昆虫细胞裂解液的抗病毒效价为3.15×104 U?ml-1。【结论】应用蜂素信号肽实现猪干扰素α在昆虫细胞上分泌表达，为临床上防治猪病毒性疫病奠定了基础。

Secreted Expression of Porcine Interferon-Alpha in Insect Cells and Its Antiviral Activity Detection

【Objective】 The objective of this study is to get porcine recombinant interferon (PoIFN)- alpha. 【Method】 Sequences derived from porcine interferon-alpha were cloned and expressed in insect cells with a C-terminal 6×Histidine tag. The authentic signal sequences of porcine interferon-alpha were substituted with the honeybee melittin signal sequences, the sequences were cloned into the baculovirus pFastBacⅠ vector of the Bac-to-Bac Baculovirus expression system. 【Result】 The recombinant proteins were successfully detected in Sf9 cells by immunofluorescence assay and in the culture supernatant by western blot analysis. The recombinant PoIFN-alpha were verified to be of high antiviral activity by inhibiting the cytopathic effect of vesicular stomatitis virus in PK-15 cells, which is about 1.07×105 U?ml-1 in supernatant and 3.05×104 U?ml-1 in insect cells, respectively. 【Conclusion】 Using the honeybee melittin signal peptides in baculovirus expression vectors are capable of secreting expression of PoIFN- alpha in insect cells. This cytokine will be explored as a poteintial therapeutic agent for porcine diseases.