RNA编辑酶ADAR1在HepG2.2.15中过表达对其上清HBsAg和HBeAg的影响#时韦美，吴佳，伍晓盼，朱席琳，刘英**5（北京协和医学院基础学院生物化学与分子生物学系，北京100005）摘要：目的：克隆人ADAR1基因的两个转录本，构建携带ADAR1基因的重组真核表达载体，瞬时转染HepG2.2.15细胞过表达，并探究其对HepG2.2.15上清HBsAg和HBeAg的影响。方法：提取Hela细胞总RNA并反转录为cDNA作为模板，分段克隆，逐步连接，将1015p110和p150全长连接到p3X-FLAG-CMV-14表达载体，双酶切和测序鉴定重组载体；将重组载体瞬时转染HepG2.2.15细胞系实现过表达后，提取细胞总RNA反转录为cDNA，通过实时荧光定量PCR检测确认过表达效果，上清除去细胞及碎片后ELISA法检测HBsAg和HBeAg水平。结果：成功构建重组载体p110-FLAG/p150-FLAG，瞬时转染HepG2.2.15细胞过表达效果良好，过表达后上清中HBsAg和HBeAg均显著上升。结论：ADAR1p110-FLAG/p150-FLAG重组载体构建成功，在HepG2.2.15细胞系过表达效果良好，过表达ADAR1p110-FLAG/p150-FLAG与HepG2.2.15细胞上清HBsAg和HBeAg的分泌相关，有利于HepG2.2.15细胞分泌HBsAg和HBeA。关键词：ADAR1；过表达；HepG2.2.15；HbsAg和HBeAg：K394.320RNAeditingenzymeADAR1overexpressedinHepG2.2.15andtheeffectonsupernatantHBsAgandHBeAgSHIWeimei,WU激a,WUXiaopan,ZHUXilin,LIUYing(NationalLaboratoryofMedicalMolecularBiology,InstituteofBasicMedicalSciendes,Chinese25303540AcademyofMedicalSciences,SchoolofBasicMedicine,PekingUnionMedicalCollege,Bei激ng100005)Abstract:Objective:ToclonethetwotrcanscriptsofADAR1(p110andp150),constuctitseukaryoticexpressionvector,andtransientlytransfectHepG2.2.15celllinewiththerecombinantvectorinordertoexploretheassociationofADAR1overexpressonandthesupernatantlevelofHBsAgandHBeAg.Methods:ThetotalRNAwasextractedfromHelacellsandthenwasreversetranscriptedintocDNA.ThefulllengthofADAR1p110/p150wasclonedandligatedtop3X-FLAG-CMV-14expressionvectorbyfragmentprogessively.TherecomibinantplasmidwasconfirmedbydoubleenzymedigestionanalysisandDNAsequencing.TransientlytransfectedHepG2.2.15cellwithADAR1p110/p150-FLAGtooverexpressADAR1,afterthattotalRNAwasextracted,cDNAwaspreparedbyRT-PCRandrealtimequantitivePCRwasperformedtoverifytheoverexpressioneffect.Finally,thelevelofHBsAgandHBeAgwasexaminedbyELISAmethod.Result:TheeukaryoticrecombinantexpressionvectorADAR1p110/p150-FLAGwassuccessfullyconstructed.WhenHepG2.2.15wastransfectedwithADAR1p110/p150-FLAG,ADAR1wasnotablyupregulatedandthesupernatantHBsAgandHBeAgweresignificantlyincreased.Conclusion:TheADAR1p110/p150-FLAGwassucessfullyconstructedandcanbeoverexpressedwellinHepG2.2.15.TheupregulationofADAR1wasassociatedwiththesecretionofHBsAgandHBeAginHepG2.2.15,stillfurther,promotethesecretionofHBsAgandHBeAg.Keywords:ADAR1;overexpression;HepG2.2.15;HBsAgandHBeAg基金项目：高等学校博士学科点专项科研基金（20101106110021）---本文来源于网络，仅供参考，勿照抄，如有侵权请联系删除---作者简介：时韦美（1990-），女，硕士，多基因疾病遗传易感性和药物遗传学研究通信联系人：刘英（1961-），女，研究员，多基因疾病遗传易感性和药物遗传学研究；单基因疾病致病基因的定位克隆.liuyingpumc@yahoo-1----本文来源于网络，仅供参考，勿照抄，如有侵权请联系删除---4550556065700引言腺嘌呤核苷脱氨酶(adenosinedeaminaseactingonRNA，ADARs)在RNA编辑过程中发挥作用，在DNA转录为RNA后使遗传信息发生改变[1]。ADAR1是ADAR家族一员，是一种特异性的修饰酶，将特殊位点上的腺苷（A）去氨基转变为次黄嘌呤（I），核糖体解码时读作鸟嘌呤（G），导致双链RNA核苷酸替换、相应蛋白氨基酸改变、RNA结构不稳定、mRNA降解、依赖RNA复制的病毒遗传...