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时间:2020-04-25
《人胶质细胞源性神经营养因子和血管内皮生长因子165双基因真核表达载体的构建与鉴定-论文.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、中国组织工程研究笫78眷第292014—07一Q9出版ChineseJournalofTissueEngineeringResearchJulygi2~O14VoL18,No.29CRTER.ong■■⋯I■●《IaentiticatIonOtplRES2·GDNF-VEG—卜■■1650b。ic。ist一roni‘ceu一karyot一一icexpressiOnvectorLiBing—nan,LiWei-dong,LinJun-tang,FengHui-genDepartmentofLifeSciencesandT
2、echno~gy,XinxiangMedicalUniversity,Xinxiang45300~HenanProvince,ChinaAbstractBACKGROUND:HumanglialcelIIine-derivedneumtrophicfactor(GDNF)andvascularendotheliaIgrowthLiBing.-nan.Ph.D.Lectumr,factor165(VEGFls5)areessentiaIgenesforcalIdiferentiation.DepartmentofLife
3、ScieflogsOBJECTIVE:ToconstructandidentifyplRES2-GDNF-VEGF165bicistroniceukaryoticexpressionvectorandTechnology,Xinxiang.METHODS:HumanGDNFgeneswereobtainedfromthegenomicDNAofhumanperipheraIbloodMedicalUniversity,XinxiangmononuclearcellsbyPCR.ThentheGDNFcDNAfragme
4、ntwasinsertedintothemultiplecloningsitesof453003,HenanProvince,plRES~-EGFP。togeneratethebicistroniceukaryoticexpressionplasmidplRES2-GDNF-EGFP.TheVEGF165ChinagenewasobtainedfromplRESz-VEGF165一EGFPplasmidbytwinPCR.ThenVEGF165cDNAfragmentwasLiWei·dong,Departmentof
5、clonedintotheplRES2-GDNF-EGFP,jnsteadofEGFP,tocreateadoublegeneco-expressingvectorplasmidLjfeSciencesandplRES2-GDNF—VEGF165containinginternalribosomeentrysites.ThenplRES2-GDNF—VEGF165wasusedtoTechnology,XinxlangtransfectHEK293cells.RT-PCRandwesternblotanalysiswe
6、reperformedtotesttheco.expressionofdoubleMedicalUniversity,Xinxianggenes.453003.HenanProvinceRESULTSANDCONCLUSION:DNAsequencinganalysisdemonstratedthaltheGDNFandVEGF,65wereChinaexactlyconsistentwiththesequencerecordedintheGenBank.Thesize"ofGDNFgenewas636bpandthe
7、sizeofVEGF16sgenewas576bp.Enzymedigestionanalysisindicatedthat,plRES2·GDNF-VEGF~e5bicistronicLiBing·nanandLiWei-dongeukaryoticexpressionvectorinsertedGDNFbandbyBglIIIBamHI.insertedIRES-VEGFls5fragmentbyBamcontributedequallytothisworkHI/NotI,andinsertedGDNF—lRES_
8、vEGF165fragmentbyBg¨I/NotI.RT.PCRandwesternblotanalysisshowedthat.afterHEK293cellsweretransfectedwithpIRES2一GDNF-VEGFIes。doublegeneswereexpressedattheCorrespondingaut
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