基于人ANP32A蛋白的C型流感病毒聚合酶纯化及PB2蛋白单克隆抗体的高效制备

Purification of influenza C virus polymerase based on hu ANP32A protein and efficient preparation of monoclonal antibody targeting PB2 protein

C型流感病毒是感染人的重要呼吸道病原,也可感染猪、狗等动物。聚合酶是C型流感病毒复制的核心,是研究病毒复制机制的重要靶标。然而目前没有商品化针对C型流感病毒聚合酶的单克隆抗体(monoclonal antibody,MAb),一定程度制约了相关研究的开展。为了制备C型流感病毒碱性聚合酶2(polymerase basic protein 2,PB2)的MAb,本研究依据人酸性核磷酸蛋白32A(human acidic nuclear phosphoprotein 32 family member A,huANP32A)与流感病毒RNA依赖性RNA聚合酶(RNA-dependent RNA polymerase,RdRp)相互作用的特性,利用免疫共沉淀技术在真核表达系统中通过融合Flag标签的huANP32A(huANP32A-Flag)纯化并富集C型流感病毒RdRp(PB1、PB2、P3),并以之作为免疫原免疫BALB/c小鼠,利用间接ELISA与Western blotting方法筛选出6株(7B11-5、8A4-5、13D9-6、8D4-1、8D4-3、9F9-4)能稳定分泌识别PB2 MAb的阳性杂交瘤细胞株。经鉴定7B11-5、8A4-5、8D4-1和8D4-3抗体亚型为IgG1型,13D9-6抗体亚型为IgG2a型,9F9-4抗体亚型为IgG3,轻链均为κ链。进一步选取1株效价高的杂交瘤细胞8D4-1来制备腹水,测定收集的小鼠腹水抗体效价为1︰64000。Western blotting结果显示,制备的MAb能够与C型流感病毒PB2发生特异性免疫反应;激光共聚焦试验结果表明,该MAb可准确检测C型流感病毒PB2的亚细胞定位。本研究通过huANP32A蛋白高效富集了C型流感病毒的RdRp,并以该复合物为抗原制备的PB2 MAb具有较好的特异性,为C型流感病毒聚合酶检测、结构分析及机制研究奠定了基础。

Influenza C virus is an important respiratory pathogen not only infecting people,but also pigs,dogs,and other animals.Polymerase is central to the replication of influenza C virus and is an important target for studying the mechanism of viral replication.However,there is no commercial monoclonal antibody(MAb)targeting influenza C virus polymerase,which hampers the development of relevant research to some extent.In order to prepare MAb targeting the polymerase basic protein 2(PB2)of influenza C virus,influenza C virus RNA-dependent RNA polymerase(RdRp,consists of PB1,PB2 and P3)was co-immunoprecipitated with Flag-tagged human acidic nuclear phosphoprotein 32A(huANP32A-Flag)from 293T cells based on the interaction between huANP32A and influenza virus RdRp.The purified RdRp was used as antigen to immunize BALB/c mice.Six positive hybridoma cell lines(7B11-5,8A4-5,13D9-6,8D4-1,8D4-3,9F9-4)that stably secrete and recognize PB2 MAb were screened by indirect ELISA and Western blotting.The subtypes of MAb 7B11-5,8A4-5,8D4-1 and 8D4-3 antibody were identified as IgG1,the subtypes of MAb 13D9-6 and 9F9-4 were IgG2a and IgG3,respectively.All the light chains of the MAbs wereκchain.A hybridoma cell line 8D4-1 with high titer was further selected to prepare ascites.The titer of mouse ascites antibody was determined to be 1:64000.Western blotting results showed that the MAb 8D4-1 had a specific immune response with ICV PB2;laser confocal assay showed that the prepared MAb 8D4-1 accurately detected the subcellular localization of PB2 subunits.Moreover,ICV RdRp was highly enriched by ANP32A.The high specific of the prepared PB2 MAb 8D4-1 may facilitate the polymerase detection,structural analysis and mechanism study of influenza C virus.