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

《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