含组氨酸蛋白标签的蓝舌病假病毒物质构建及定量分析

Abstract

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

CLC Number:

S855.3

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

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