作　　者： ; ; ; ; ;

机构地区： 中国检验检疫科学研究院

出　　处： 《中国兽医科学》 2014年第10期997-1002,共6页

摘　　要： 为研制一种无生物传染性且极易保存稳定的小反刍兽疫病毒(PPRV)RNA阳性质控品,以pTrcHis-MS2质粒为模板,借助点突变技术在噬菌体MS2编码的外壳蛋白第15位与第16位氨基酸基因之间插入组氨酸蛋白标签的相应序列,构建了pTrcMS载体。将pGEM-T-N382质粒与pTrcMS质粒分别进行KpnⅠ+HindⅢ双酶切、连接从而构建了pTrcMS-PPRV重组质粒。将pTrcMS-PPRV重组菌进行原核表达,借助偶联组氨酸蛋白标签的磁珠捕获技术获得纯化的小反刍兽疫病毒样颗粒,对它进行特性鉴定并定量检测。结果显示,pTrcMS-PPRV病毒样物质纯度高;电镜下为不规则的、直径约为26nm的多边形;耐RNA酶A,稳定;经荧光RT-PCR定量检测,此病毒样物质的溶液浓度相当于108.56 PFU/mL。本研究将为小反刍兽疫病毒分子生物学的检测提供阳性质控物质。 An uninfectious and steady quality control of peste des petits ruminants virus(PPRV)RNA is vital.pTrcMS vector was constructed,of which pTrcHis-MS2 plasmid was introduced 6His-tags between codons 15 and 16of MS2 bacteriophage coat protein using a site-directed mutagenesis method.pGEM-TN382 plasmid was digested with KpnⅠ+HindⅢand ligated into KpnⅠ+HindⅢsites in pTrcMS,creating the recombinant plasmid pTrcMS-PPRV.pTrcMS-PPRV in Escherichia coli was induced with 0.5mmol/L IPTG,then the Armord-RNA(AR)particles were captured with MagneHis Ni-Particles from bacterial lysates,and verified by real-time RT-PCR method.The His-tagged AR particles were highly pure.A TEM photograph showed that the AR particles had the shape of a round particle that was 26 nm in diameter.The rarefied AR particles were able to survive RNase A and extremely stable.The concentration of the virus-like particles(VLPs)was equivalent to 108.56 PFU/mL by real-time PCR.The His-tagged AR particles will provide quality-control for PPRV molecular detection.

关 键 词： 小反刍兽疫 病毒样颗粒 组氨酸蛋白标签 纯化 定量分析

领　　域： [农业科学] [农业科学] [农业科学]