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1、猪AP0BEC3F基因克隆及真核表达载体的构建与鉴定孙宇二罗佳；马玉媛章金刚1(100850北京军爭医学科学院野战输血研究I；解放军第305医院检验科2；广西大学动物科学技术学院彳)［摘要］目的克隆猪载脂蛋白BmRNA编辑酶催化多肽样蛋A3F(apolipoproteinBniRNAeditingenzyme,catalyticpolypeptide3F,APOBEC3F)基因，构建真核表达载体实现其在体外表达与鉴定。方法分离21周龄，体重在25〜30公斤的雌性健康五指山猪4头的外周血单个核细胞，TRIZOL法提取细胞总RNA,RT-PCR技术特
2、界性扩增目的基因，B的基因插入真核表达载体pDSredl-Nl及改造的pCDNA3-Flage载体构建农达质粒并在PK-15细胞小进行表达。激光共聚焦显微镜及Westernblot方法对目的基因的表达进行鉴定。结果克隆的目的基因与公布的基因序列同源性达99%；激光共聚焦显微镜显示目的基因在PK-15细胞中实现了表达，且表达产物定位于细胞质内；Westernblot鉴定结果表明，表达的H的蛋白大小与预期相符。结论成功构建了猪APOBEC3F真核表达载体，为进一步研究其与猪内源性反转录病毒(Porcineendogenousretrovirus,PE
3、RV)和互作用奠定了基础。［关键词1APOBEC3F；克隆；真核表达载体；鉴定ThecloningofPorcineAPOBEC3FgeneandtheconstructionandidentiflcationofeukaryoticexpressionvectorsSunYu12,MaYuyuan1,LuoJia3,ZhangJingang^'instituteofTransfusionMedicine,AcademyofMilitaryMedicalSciences,Beijing100085:2The305HospitalofPLALabo
4、ratoryDepartment,Beijing100017；'CollegeofAnimalsSciences&Technology,GuangxiUniversity,Nanning530005・)［Abstract］ObjectiveToclonethegeneofporcineapolipoproteinBmRNAeditingenzyme,catalyticpolypeptide-like3F(APOBEC3F)andconstmcttheeukaryoticexpressionvectorfortheexpressionandiden
5、tificationinvitro・MethodsSeparatingthePeripheralbloodmononuclearcells(PBMCs)fromthebloodsoffourfemalehealthyWuzhishanporcinewhichageswere21weeksandweightwere25〜30kg,thenextractingtheRNAfromtheporcinePBMCbytheTRAZOregent.ThecodingregionofAPOBEC3Fgenewasampl讦iedbyRT-PCR,andthen
6、thegenefragmentwasinsertedintotherecombinanteukaryoticexpressionvectorpDSredl-NlandpCDNA3-Flage-pA3FwhichhadbeentransformedtoconstructtheeukaryoticexpressionplasmidsofpDSred1-N1-pA3FandpCDNA3-Flage-pA3F,whichweretransintoPK-15cellbyLip2000transfectionagent,respectively.Theexp
7、ressedproductsinthepk-15cellsweredetectedbyFluorescencedetectionbytheLaserscanningconfocalmicroscopeandWesternblot.ResultsTheHomologousofthegenewhichhadbeenclonedwas99%comparetothatpublishedonline・TheLaserscanningconfocalmicroscopedetectedtheredfluorescenceoftheexpressedfusio
8、nproteinofthepA3Fandredfluorescentprotein(RFP)andthatlocatedinthecyt
