流感病毒RNA聚合酶串联亲和纯化方法的建立  

Tandem affinity purification of influenza virus RNA polymerase

作　　者：李印[1] 邓国华[1] 崔佳莹[1] 于洋[2] 丁晴薇[3] 陈化兰[1] 

机构地区：[1]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,黑龙江哈尔滨150001 [2]华南农业大学,广东广州510642 [3]湖南农业大学,湖南长沙410127

出　　处：《中国预防兽医学报》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)

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.

关 键 词：H5亚型禽流感病毒 RNA聚合酶复合体 串联亲和纯化 

分 类 号：S852.65[农业科学—基础兽医学]