| 猪α干扰素在杆状病毒中表达及其对PRRSV的抑制作用 |
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| 引用本文： | 王彦彬,黄艳群,崔保安,陈红英,王亚宾,张红英.猪α干扰素在杆状病毒中表达及其对PRRSV的抑制作用[J].中国兽医学报,2009,29(12). |
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| 作者姓名： | 王彦彬 黄艳群 崔保安 陈红英 王亚宾 张红英 |
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| 作者单位： | 河南农业大学,牧医工程学院,河南省动物性食品安全重点实验室,河南,郑州,450002 |
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| 基金项目： | 国家科技支撵计划专项基金，郑州市创新型科技人才队伍建设工程基金 |
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| 摘 要： | 猪α干扰素具有抗病毒作用,临床上具有广阔的应用前景.为获得重组猪α干扰素,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α干扰素基因插入供体质粒pFastBac~(TM) Ⅰ多克隆位点,置于pH启动子控制下,在C端融合6个组氨酸标签以利于纯化.将重组转移栽体质粒转化DH10感受态细胞获得重组穿梭质粒rBacmid,转染对数生长期的Sf9昆虫细胞获得重组杆状病毒.重组蛋白通过间接免疫荧光、Western-blotting证明在重组杆状病毒感染的昆虫细胞中获得表达.镍亲和层析柱纯化的重组蛋白经SDS-PAGE电泳相对分子质量为19 000.通过在猪肾细胞(PK-15)上抑制猪水泡性口炎病毒(VSV)致病变作用检测其抗病毒活性为9.67×10~4 U/mL.昆虫培养上清及细胞裂解液经2~8稀释在Mare-145细胞上能够抑制猪蓝耳病病毒增殖.从而为进一步作为抗病毒药物应用于猪疫病的防治研究奠定了基础.
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| 关 键 词： | 猪 干扰素 杆状病毒 表达 抑制作用 猪繁殖与呼吸综合征病毒 |
Expression of porcine interferon-alpha using a baculovirus and its inhibition activity on the replication of PRRSV |
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| WANG Yan-Bin,HUANG Yan-qun,CUI Bao-an,CHEN Hong-ying,WANG Ya-bin,ZHANG Hong-ying.Expression of porcine interferon-alpha using a baculovirus and its inhibition activity on the replication of PRRSV[J].Chinese Journal of Veterinary Science,2009,29(12). |
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| Authors: | WANG Yan-Bin HUANG Yan-qun CUI Bao-an CHEN Hong-ying WANG Ya-bin ZHANG Hong-ying |
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| Abstract: | The gene sequences encoding mature porcine interfeton-alpha(PoIFN-α)fused with a C-terminal 6×Histidine tag were cloned into the baculovirus pFastBac~(TM) I vector of the Bac-to-Bac baculovirus expression system.and expressed in insect cells.The recombinant proteins were detected in expressing cells by immunofluorescence assay and Western-blotting analysis.The rPoIFN-α by purified nickel affinity spin column was a 19 000 protein as confirmed by SDS-PAGE.The recombinant PoIFN-α was of cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in PK-15 cells,which is about 9.67×10~4 U/mL.The dilution of 2~8 of rPoIFN-α in supernant and insect cells could protect the Marc-145 from the cytopathic effect cause by 100TCID50 of porcine reproductive and respiratory syndrome virus.The results will allow US to explore this cytokine as a poteintial therapeutic agent for porcine diseases. |
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| Keywords: | porcine interferon baculovirus expression inhibition activity PRRSV |
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