人BTC和PDX1基因的克隆及表达

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1、上海交通大学硕士学位论文人BTC和PDX-1基因的克隆及表达姓名：陈寒蓓申请学位级别：硕士专业：内科学内分泌与代谢病指导教师：苏青20070501上海交通大学医学院2004级硕士学位论文结论：重组人成熟BTC蛋白在pET32a（+）表达系统中得到高效表达，所纯化蛋白具有促进NIH3T3细胞体外增殖的作用。成功构建了pcDNA3.1-PDX-1重组真核表达载体，并在NIH3T3细胞中表达。关键词：BTC，PDX-1，基因克隆，表达，蛋白质纯化，转染上海交通大学医学院2004级硕士学位论文CLONINGANDEXPRESSIONOFHUMANBTCGE

2、NEANDPDX-1GENEABSTRACTBTCgeneandPDX-1geneplayanimportantroleinthedifferen-tiationofpancreaticßcell.Inthestudy,wecloneandexpresshumanBTCgeneandPDX-1gene.ObjectiveToobtainabundanthumanbetacellulinwithbiologicalactivity.ToconstructaeukaryoticexpressionvectorofhumanPDX-1gene,andto

3、detectitsexpressioninNIH3T3celllines.MethodsThewholematureproteincodingsequenceofBTCgeneandthewholecodingsequenceofPDX-1genewereamplifiedbypolymerasechainreaction(PCR)methodappliedtohumanpancreaticß-celltumorscDNA.ThefragmentswereclonedintoprokaryoticexpressionvectorpET32a(+)p

4、lasmidandeukaryoticexpressionvectorpcDNA3.1plasmid.ThepET32a(+)-BTCwastransformedintoE.coli上海交通大学医学院2004级硕士学位论文BL21andthefusionproteinwasexpressedunderIPTG.Thefusion2+proteinwaspurifiedbyNiaffinitychromatography.SDS-PAGEandWesternblotwereusedtodetermineexpressionandpurificatio

5、noftheexpectedprotein.BTCwasaddedtocultureNIH3T3cellstostudybiologicalactivity.ThepcDNA3.1-PDX-1wasverifiedbydoublediges-tionandDNAsequencing.TheexpressionofPDX-1geneinNIH3T3cellswasassayedbyWesternblot.ResultsLotsofBTCfusionproteinwereproduced.Thepurifiedproteincanstimulateth

6、eproliferationofNIH3T3cells.PDX-1genehadbeenclonedintopcDNA3.1vectorcorrectly.WesternblotshowedPDX-1genewasexpressed,whichwasdetected24hafterpcDNA3.1-PDX-1plasmidwastransfectedintoNIH3T3cells.ConclusionThehumanbetacellulincanbesuccessfullyobtainedfromthepET32a(+)systermwithbio

7、logicalactivityofstimulatingtheproliferationofNIH3T3cells.TherecombinanteukaryonexpressionvectorpcDNA3.1-PDX-1wassuccessfullyconstructedandexpressedintheNIH3T3celllines.KEYWORDS:BTC,PDX-1,geneclone,expression,purifedprotein,transfection上海交通大学学位论文原创性声明本人郑重声明：所呈交的学位论文，是本人在导师的指导下

8、，独立进行研究工作所取得的成果。除文中已经注明引用的内容外，本论文不包含任何其他个人或集体已经发表或撰写过的作品成果。对本