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《B7-H1启动子荧光素酶报告基因载体的构建及活性检测.pdf》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
1、2014年7月基础医学与临床July2014第34卷第7期Basic&ClinicalMedicineVo1.34No.7文章编号:1001—6325(2014)07—0955.06研究~g-文B7一H1启动子荧光素酶报告基因载体的构建及活性检测吴胜昔,陈永文,朱广倍,费蕾,吴玉章(1.重庆理工大学药学与生物工程学院,重庆400054;2.第三军医大学基础部免疫学教研室,重庆400017)摘要:目的构建含B7.H1启动子的荧光素酶报告基因载体,转染肝癌HepG2细胞进行活性检测。方法PCR扩增B7-H1启动子上游区域基因片段,并插入到含有荧光素酶报告基
2、因的载体PGL3一Basic中构建重组子,经双酶切和测序鉴定后,将重组子转染HepG2细胞,用双荧光素酶报告基因检测系统检测荧光素酶表达活性。结果成功地构建了6种不同长度的PGL3-B7一H1.Luc荧光素酶表达质粒,分别命名为P88、P175、P203、P398、P723和P960。瞬时转染HepG2后经报告基因检测,P175、P203、P398、P723和P960所含5段启动子均具有转录活性,IFN-作用后启动子活性明显增强,而P88转染组IFN一作用后,荧光素酶活性无显著变化。结论成功构建了B7一H1启动子的荧光素酶报告基因载体。关键词:B7H1
3、;启动子;双荧光素酶报告基因检测;活性测定中图分类号:R392文献标志码:AConstructionofluciferasereportergenevectorscontainingB7-H1promoterregionandpromoteractivityassayWUSheng—xi,CHENYong—wen,ZHUGuang—bei,FEILei,WUYu.zhang(1.SchoolofPharmacyandBioengineering,ChongqingUniversityofTechnology,Chongqing400054;2.Dept
4、.ofImmunology,theThirdMilitaryMedicalUniversity,Chongqing400017,China)Abstract:ObjectiveToconstructluciferasereportergenevectorscontainingB7一H1promoterregionandtodetectpromoteractivityinHepG2cells.MethodsFragmentsoftheB7一H1gene5-UTRpromoterswereamplifiedbyPCRandclonedintoPGL3bas
5、icvectorandnamedasP88,P175,P203,P398,P723,andP960respectively.ThenHepG2cells,weretransfectedwiththeseconstructedplasmids.TheexpressionofluciferasebeforeandaftertheadditionofIFN一^ywasdetectedbydual—lueiferase(LUC)reporterassaysystemkit.ResultsFiveluciferasere.portergenevectorscon
6、tainingB7一H1promoterregionweresuccessfullyconstructed.TherecombinantconstructsweretransientlytransfeetedintoHepG2cells.AftertheadditionofIFN一^y,LUCdetectionassayshowedthattheex—pressionofLUCwassignificantlyenhancedbyP175,P203,P398,P723orP960,butnotbyP88.ConclusionsLueiferaserepo
7、rtergenevectorscontainingB7-H1promoterregionweresuccessfullyconstructed,whichmaybeval—uableforfurtherstudyonB7一H1promoteractivityandmolecularmechanism.Keywords:B7-H1;promoter;dua1.1uciferaserepo~erassaysystem;activityassay收稿日期:2014—02—28修回日期:2014.04.17基金项目:重庆市基础与前沿研究计划(cstc2013j
8、cyjA10068);重庆市教委科学技术研究项目(KJ130802)‘通信作者(corresp
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