人肝癌血管内皮生长因子受体3逆转录病毒载体构建

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1、人肝癌血管内皮生长因子受体3逆转录病毒载体构建魏春山1童光东1(通讯作者｝王宏艳2(1广州中医药大学深圳附属医院<深圳市中医院>广东深圳518033)(2华中科技大学同济医学院附属同济医院湖北武汉430074)【】目的构建在人肝癌HePG2细胞内可稳定表达血管内皮生长因子受体3(VEGFR3)的逆转录病毒载体。方法采用PCR技术扩增VEGFR3片段，克隆至pBABE-puro质粒,构建重组质粒pBABE-puro-VEGFR3,经PCR、酶切、测序等验证后，体外与PIK包装质粒共同转染293FT细胞

2、，获得携带VEGFR3基因的重组逆转录病毒，感染人肝癌HepG2细胞，获得转入VEGFR3的HepG2细胞，通过QPCR和Westernblot进一步验证VEGFR3在HepG2细胞内的表达。结果①获得携带VEGFR3基因的重组质粒pBABE-puro-VEGFR3和人肝癌细胞株HepG2-pBABE-puro-VEGFR3；②QPCR和Westernblot分别检测到VEGFR3在靶细胞HepG2-pBABE-puro-VEGFR3中的稳定表达。结论成功构建了携带VEGFR3基因的人肝癌细胞重组逆转录病毒载体

3、。【关键词】血管内皮生长因子受体3逆转录病毒载体HepG2细胞【】R730【】A【】2095-1752(2014)17-0049-04ConstructionofaRetroviralVectorofHepG2CellLineCarryingVascularEndothelialGrowthFactorReceptor-3TONGGuang-dongl,WEIChun-shanl,WANGHong-yan21ShenzhenhospitalaffiliatedtoGuangzhouuniversityofChi

4、nesemedicine(ShenzhentraditionalChinesemedicinehospital),Shenzhen,518033,Guangdongprovince2TongjihospitalaffiliatedtoTongjimedicalcollegeofHuazhonguniversityofscience&technology,Wuhan,430074,Hubeiprovince［Abstract］ObjectiveToconstructaretroviralvectorcar

5、ryingvascularendothelialgrowthfactorreceptor-3(VEGFR3)andtransfectVEGFR3intoHepG2cells.MethodsVEGFR3genewasamplifiedbyPCRandsubclonedintotheretroviralvectorpBABE-purotoconstructaretroviralplasmid(pBABE-puro-VEGFR3)expressingVEGFR3.TheVEGFR3geneinsertwasteste

6、dbyPCR,restrictionenzymedigestion,andDNAsequencing.TherecombinantretrovirusescarryingVEGFR3wereproducedin293FTcellsco-transfectedwithpBABE-puro-VEGFR3andthepackagingplasmidsPIK,andtheninfectedHepG2cells.TheVEGFR3mRNAandproteinexpressionsweredeterminedbythere

7、versetranscription-PCRandWesternblotting,respectively.ResultsTheretroviralplasmidcarryingVEGFR3(pBABE-puro-VEGFR3)wasconstructedsuccessfully.TheexpressionsofVEGFR3mRNAandVEGFR3proteininhepG2cellsafterdeliveredVEGFR3intopBABE-puro-VEGFR3couldbedetectedbyQPCRa

8、ndWesternblot.ConclusionTherecombinantretrovirusvectorhepG2-pBABE-puro-VEGFR3hasbeensuccessfullyconstructed,whichcanstableexpressVEGFR3protein.【Keywords】VEGFR3retrovirusvectorHepG2cell血管和淋巴在肿瘤生长