猪干扰素α和γ在杆状病毒中共表达及对PRRSV抑制作用

doi:10.3864/j.issn.0578-1752.2011.09.021

摘要/Abstract

摘要： 【目的】制备复合型猪干扰素α和γ进行应用抗病毒研究。【方法】本研究应用Bac-to-Bac杆状病毒/昆虫细胞表达系统,将编码猪干扰素α和γ成熟蛋白基因插入供体质粒pFastBacDual，分别置于PH和P10启动子控制下，引入蜂素信号肽（honeybee melittin signal peptide, HBM）基因取代猪干扰素α和γ原有信号肽基因以实现分泌型表达，并在C端分别融合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，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。重组杆状病毒感染昆虫细胞，鉴定重组蛋白的活性。【结果】通过间接免疫荧光、Western-blot证明猪干扰素α和γ重组蛋白在重组杆状病毒感染的昆虫细胞中均获得表达，表达产物主要分布在培养上清中。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3.24×107 U·mL-1。在Marc-145细胞，昆虫细胞上清经2-11稀释能够抑制100个TCID50的PRRSV的致细胞病变作用。【结论】应用杆状病毒表达系统实现猪干扰素α和γ在昆虫细胞上分泌共表达，重组蛋白在细胞上对PRRSV具有抑制作用。

关键词: 猪 , 干扰素 , 杆状病毒 , 表达 , 繁殖与呼吸综合征病毒

Abstract: 【Objective】Compound recombinant porcine interferon α and interferon γ were prepared for clinical antiviral investigation 【Method】 Nucleotide sequences encoded mature porcine interferon-alpha and interferon-gamma were cloned and inserted into pFastBacDual under the control of PH promoter and P10 promoter, respectively. The authentic signal sequences of porcine interferon-alpha and interferon-gamma were substituted with the honeybee melittin signal sequence, and fused with C-terminal 6×Histidine tag for easy purification. The constructed plasmid was transformed into DH10 competent cells for homologous recombination, the recombinant shuttle plasmid Bacmid then transfected to Sf9 insect cells to obtain recombinant baculovirus. After infection with recombinant baculovirus, the recombinant protein activity was detected.【Result】The recombinant proteins were successfully detected in Sf9 cells by immunofluorescence assay and Western blot analysis. The expression products were mainly detected in the culture medium. The compound recombinant interferon-α & γ were verified to be of high antiviral activity by inhibiting the cytopathic effect of vesicular stomatitis virus in PK-15 cells, which is about 3.24×107 U·mL-1 in supernatant. The results also suggest that in vitro rpIFN-α & γ protected Marc-145 from cytopathic effect by PRRSV of 100 TCID50. 【Conclusion】 The baculovirus expression system to achieve porcine interferon-α and γ were successfully secreted in insect cells, the recombinant protein in cells inhibited the replication of PRRSV.

Key words: porcine , interferon , baculovirus , expression , PRRSV

王彦彬，孙向丽，魏战勇，陈红英，杨明凡，崔保安. 猪干扰素α和γ在杆状病毒中共表达及对PRRSV抑制作用[J]. 中国农业科学, 2011, 44(9): 1931-1938.

WANG Yan-bin, SUN Xiang-li, WEI Zhan-yong, CHEN Hong-ying, YANG Ming-fan, CUI Bao-an. Co-Expression of Porcine Interferon-Alpha and Interferon-Gamma Using a Baculovirus and Its Inhibitant Activity on PRRSV[J]. Scientia Agricultura Sinica, 2011, 44(9): 1931-1938.

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