流感病毒RNA聚合酶串联亲和纯化方法的建立

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(H5Nl) 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-35PBl 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.