ebf3与egfp融合蛋白表达载体的构建及其在hepg2中的表达

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1、EBF3与EGFP融合蛋白表达载体的构建及其在HepG2中的表达作者：毛丽萍王惠民,王跃国,汪晓莺【摘要】目的:构建早期B细胞因子3（EBF3）与增强型绿色荧光蛋白（EGFP）的融合基因真核表达载体pEGFP/EBF3,使EBF3EGFP融合蛋白在人肝癌细胞株HepG2中得到表达。方法:采用RTPCR技术,从人胎盘组织总RNA中逆转录并扩增出EBF3全长编码基因,构建EBF3与EGFP的融合基因真核表达载体pEGFP/EBF3,用脂质体转染技术将pEGFP/EBF3导入HepG2,使EBF3EGFP融合蛋白得到表达。结果:经转化细菌、抽提质粒、酶切鉴定和DNA序列分析

2、证实,EBF3基因已正确插入pEGFPN1中EGFP基因的上游,获得融合基因表达载体pEGFP/EBF3。将pEGFP/EBF3导入HepG2细胞24h后,在倒置荧光显微镜下可观察到EBF3EGFP融合蛋白主要表达于核内,转染pEGFP/EBF3和pEGFPN1后48h的转染效率分别为52%和59%。Westernblot证实转染pEGFP/EBF3后24h和48h细胞质和细胞核中均检出相对分子质量（Mr）为87000的EBF3EGFP融合蛋白,转染pEGFP/EBF3后48h和72h,S期细胞比例均明显高于pEGFPN1转染细胞组和未转染细胞组,表明EBF3基因

3、的导入可诱导细胞从G1期向G2期发展,从而促进细胞增殖。结论:成功构建了真核表达载体pEGFP/EBF3,并在HepG2细胞中进行了表达,为进一步研究EBF3的功能提供实验基础。14【关键词】早期B细胞因子3;融合蛋白;增强型绿色荧光蛋白;HepG2细胞［Abstract］AIM:ToconstructtheeukaryoticexpressionvectorforearlyhumanBcellfactor3(EBF3)fusedwiththeenhancedgreenfluorescentprotein(EGFP)andexpressthefusionproteinEB

4、F3EGFPinHepG2cells.METHODS:TheintactEBF3genewasamplifiedfromRNAisolatedfromtheplacentaltissuebyRTPCR.ThenitwasinsertedintothepEGFPN1vectortoconstructtherecombinanteukaryoticexpressionvectorpEGFP/EBF3.ThefusionproteinEBF3EGFPwasexpressedinHepG2cellsbythetransfectionofpEGFP/EBF3intothece

5、lls.RESULTS:ThepEGFP/EBF3recombinantexpressionvectorwasconstructedandconfirmedbyDNAsequencing,enzymaticdigestionandPCRidentification.24hafterthefusionproteinEBF3EGFPwastransfectedwihtpEGFP/EBF3,itwasobservedmainlyinthecellularnucleusundertheinvertedfluorescencemicroscope.BothpEGFP/EBF3and

6、pEGFPN1weretransfectedintohumanHepG2cells.24hafterthetransfection,theproportionoftransfectionwasabout52%and59%,respectively.WesternblotanalysisconfirmedthattheEBF3EGFPfusionproteinsofMr8714000weredetectedinthecytoplasmicandnuclearproteinofHepG2transfectedwithpEGFP/EBF3for24hor48h.Thecell

7、proportioninSphaseincreasedinHepG2cellstransfectedwithpEGFP/EBF3incomparisonwithHepG2cellstransfectedwithpEGFPN1oruntransfected.ThesefindingssuggestedthatthetransfectionofEBF3geneintoHepG2inducedthecellproliferationfromG1phasetoG2phasebyincreasingthenumberofc