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流感病毒RNA聚合酶串联亲和纯化方法的建立 被引量：2

Tandem affinity purification of influenza virus RNA polymerase
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摘要 为建立流感病毒RNA聚合酶的串联亲和纯化方法,本实验由鸡胚尿囊液中提取病毒核酸,经过RT-PCR和常规PCR方法扩增禽流感病毒株A/Duck/Guangxi/35/01(H5N1)的PB1、PB2和PA的cDNA。分别将PB2基因连接于pcDNATAP载体中,PB1和PA基因连接于pcDNA3A载体中构建了pcDNA-35PB2TAP、pcDNA-35PB1和pcDNA-35PA重组质粒。将构建的真核表达重组质粒共转染293T细胞,经过串联亲和纯化方法获得流感病毒依赖RNA的RNA聚合酶复合体(RdRp)。利用RNA体外合成试验验证获得的RdRp具有生物学活性。 To express and purify the influenza virus RNA polymerase,the total RNA of H5 subtype avian influenza strain A/Duck/Guangxi/35/01（H5N1） was extracted from allantoic fluid and the genes of PB1,PB2 and PA were amplified by RT-PCR.The recombinant plasmids of pcDNA-35PB2TAP,pcDNA-35PB1 and pcDNA-35PA were constructed based an eurokaryotic expression vetors of pcDNATAP and pcDNA3A,respectively.Furthermore,the RNA-dependent RNA polymerase complex（RdRp） of the virus expressed in the co-transfected 293T with those recombinant plasmids and purified by tandem affinity purification（TAP）.RNA synthesized assay showed that the purified RdRp possessed biological activities.
作者 李印 邓国华 崔佳莹 于洋 丁晴薇 陈化兰
机构地区 中国农业科学院哈尔滨兽医研究所农业部动物流感重点开放实验室/兽医生物技术国家重点实验室 华南农业大学 湖南农业大学
出处 《中国预防兽医学报》 CAS CSCD 北大核心 2011年第11期841-844,共4页 Chinese Journal of Preventive Veterinary Medicine
基金 国家十五重点科技攻关课题(2004BA519A054)
关键词 H5亚型禽流感病毒 RNA聚合酶复合体 串联亲和纯化 H5 subtype avian influenza virus RNA polymerase tandem affinity purification
分类号 S852.65 [农业科学—基础兽医学]
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参考文献14

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二级参考文献52

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共引文献18

同被引文献15

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中国预防兽医学报
2011年 第11期

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