含组氨酸蛋白标签的蓝舌病假病毒物质构建及定量分析

中国畜牧兽医 ›› 2014, Vol. 41 ›› Issue (9): 68-73.

含组氨酸蛋白标签的蓝舌病假病毒物质构建及定量分析

邓俊花, 林祥梅, 张永宁, 王彩霞, 吴绍强

中国检验检疫科学研究院, 北京 100029

收稿日期:2014-03-04 出版日期:2014-09-20 发布日期:2014-09-24
通讯作者: 吴绍强 E-mail:sqwu@sina.com
作者简介:邓俊花（1980-），女，山东人，硕士，助理研究员，研究方向：动物检疫。
基金资助:
国家科技支撑计划项目（2013BAD12B01）；国家质检总局科研计划项目（2012IK026）。

Construction and Quantitative Analysis of His-tagged Bluetongue Virus-like Particles

DENG Jun-hua, LIN Xiang-mei, ZHANG Yong-ning, WANG Cai-xia, WU Shao-qiang

Chinese Academy of Inspection and Quarantine, Beijing 100029, China

Received:2014-03-04 Online:2014-09-20 Published:2014-09-24

摘要/Abstract

摘要： 基于蓝舌病（bluetongue，BT）的危害及RNA阳性质控品研制的重要性，试验将pTrcHis-MS2质粒借助点突变技术在噬菌体外壳蛋白15和16氨基酸之间插入组氨酸（His）蛋白标签，构建了pTrcMS载体。将pMD19-T-BTV质粒与pTrcMS质粒分别进行KpnⅠ和Hind Ⅲ双酶切，然后连接构建pTrcMS-BTV重组质粒。将pTrcMS-BTV重组菌进行原核表达，产物借助偶联His蛋白标签的磁珠捕获技术获得纯化的蓝舌病假病毒颗粒，进行特性鉴定并定量。结果显示，研制的pTrcMS-BTV物质经PCR方法评价纯度高，无基因DNA污染；透射电镜观察形态为不规则的直径约为26 nm的多边形；RT-PCR方法检测稳定性表明该物质耐RNase A，易保存；应用荧光RT-PCR检测表明该物质溶液最低检测限可达到2.5×10²拷贝/mL。蓝舌病假病毒物质将为蓝舌病分子生物学检测提供阳性质控物质。

关键词: 蓝舌病; 假病毒物质; 组氨酸蛋白标签; 纯化; 定量

Abstract: In view of the harm of bluetongue (BT) and the importance of RNA quality control,pTrcMS vector was constructed by which p-MS2 plasmid was introduced 6His-tags between codons 15 and 16 of MS2 bacteriophage coat protein (CP), using a site-directed mutagenesis method.pMD19-T-BTV plasmid was digested with KpnⅠ/HindⅢ and ligated into pTrcMS to create the recombinant plasmid pTrcMS-BTV.pTrcMS-BTV in E.coli was induced with 0.5 mmol/L IPTG,then the Armord-RNA(AR) particles were captured with MagneHis^TMNi-Particles from bacterial lysates,and verified by Real-time RT-PCR method.The PCR results indicated that the His-tagged AR particles were highly pure without genome DNA.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 withstand RNase A and extremely stable.The limit of detection was determined to be 2.5×10² copies/mL by Real-time PCR.The His-tagged AR particles would provide quality-control for BT molecular detection.

Key words: bluetongue; virus-like particles (VLPs); 6His-tags; purification; quantitative

中图分类号:

S855.3

邓俊花, 林祥梅, 张永宁, 王彩霞, 吴绍强. 含组氨酸蛋白标签的蓝舌病假病毒物质构建及定量分析[J]. 中国畜牧兽医, 2014, 41(9): 68-73.

DENG Jun-hua, LIN Xiang-mei, ZHANG Yong-ning, WANG Cai-xia, WU Shao-qiang. Construction and Quantitative Analysis of His-tagged Bluetongue Virus-like Particles[J]. , 2014, 41(9): 68-73.

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