Wang Yan-Bin, Wang Zhen-Ya, Chen Hong-Ying, Cui Bao-An, Wang Ya-Bin, Zhang Hong-Ying, Wang Rui
Key Laboratory of Animal-derived Food Safety of Henan Province, College of Husbandry and Veterinary, Henan Agricultural University, Zhengzhou, Henan 450002, China.
Vet Immunol Immunopathol. 2009 Dec 15;132(2-4):314-7. doi: 10.1016/j.vetimm.2009.05.017. Epub 2009 Jun 6.
The gene sequence encoding mature porcine interferon-gamma (PoIFN-gamma) fused with a C-terminal 6x histidine tag was cloned into the baculovirus pFastBac Dual vector of the Bac-to-Bac Baculovirus expression system under the control of PH promoter. The authentic signal sequence of porcine interferon-gamma was substituted with the honeybee melittin (HBM) signal sequence, and expressed in insect cells. The recombinant proteins were detected by SDS-PAGE and immunofluorescence assay. The nickel affinity column purified recombinant porcine interferon-gamma with HBM signal peptide (rPoIFN-gammaH) was shown to be a 19kDa protein as confirmed by Western blot analysis. The recombinant PoIFN-gammaH was shown to have cytokine activity, inhibiting the cytopathic effect of vesicular stomatitis virus (VSV) in PK-15 cells at about 1.07x10(6)U/mL. The 2(-7) dilution of the rPoIFN-gammaH in culture supernatant protected the MARC-145 cells from the cytopathic effect caused by 100TCID(50) of porcine reproductive and respiratory syndrome virus.
将编码成熟猪干扰素-γ（PoIFN-γ）并融合C末端6x组氨酸标签的基因序列，在PH启动子的控制下克隆到杆状病毒表达系统的杆状病毒pFastBac Dual载体中。用蜜蜂蜂毒肽（HBM）信号序列替换猪干扰素-γ的真实信号序列，并在昆虫细胞中表达。通过SDS-PAGE和免疫荧光测定法检测重组蛋白。经蛋白质免疫印迹分析证实，用镍亲和柱纯化的带有HBM信号肽的重组猪干扰素-γ（rPoIFN-γH）是一种19kDa的蛋白质。重组PoIFN-γH具有细胞因子活性，在PK-15细胞中抑制水疱性口炎病毒（VSV）的细胞病变效应，效价约为1.07x10(6)U/mL。培养上清液中rPoIFN-γH的2(-7)稀释度可保护MARC-145细胞免受100TCID(50)猪繁殖与呼吸综合征病毒引起的细胞病变效应。
