人cd44基因真核表达载体构建及在乳腺癌细胞中的表达

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1、人CD44基因真核表达载体构建及在乳腺癌细胞中的表达作者：房新建许文林张徐钱晖陈琛方莉莉陈巧云【摘要】目的：从人类乳腺癌耐多柔比星细胞株细胞（MCF7/Adr)中克隆CD44基因并构建CD44基因真核表达载体pcDNA3.1CD44;将pcNDA3.1CD44稳定转染入人乳腺癌细胞株（MCF7）细胞中。方法：从MCF7/Adr细胞中提取总RNA进行RTPCR，获得全长的cDNA模板；将含有CD44基因的cDNA模板使用限制性内切酶进行双酶切获得带有酶切位点的CD44基因,再将带有酶切位点的CD4

2、4基因经TA克隆后测序验证，然后亚克隆入真核表达载体pcDNA3.1中，双酶切鉴定后再次测序验证。脂质体法将pcDNA3.1CD44质粒转染到MCF7细胞中，48小时后RTPCR和流式细胞术检测转染前后CD44基因、蛋白在MCF7细胞的表达变化。结果：成功从人MCF7/Adr细胞中克隆获得CD44基因并构建真核表达载体，转染后在MCF7中检测到CD44基因mRNA和蛋白水平表达上调；将上述表达CD44基因及蛋白的瞬时转染MCF7细胞经G418筛选两周后获得阳性克隆。结论：成功克隆和建立人CD4

3、4基因真核表达质粒；pcDNA3.1CD44能在MCF7细胞中表达；在MCF7/Adr中新发现一种CD44基因的变异体(基因库号FJ216964)；这为进一步研究其基因功能打下了基础。【关键词】CD44；多药耐药；侵袭；乳腺癌15［Abstract］Objective:TocloneCD44genefrommultidrugresistanthumanmammarycarcinomacells(MCF7/Adr)andconstructitseukaryoticexpressionvector.Wh

4、ichwastransfectedintoMCF7cellstoobtainpositionclone.Methods：ThefulllengthcDNAencodingCD44wasobtainedbyRTPCRfromthetotalRNAisolatedfromtheMCF7/AdrcellsandclonedintopMD19Tvector.TheCD44genesequenceandreadingframewereconfirmedbytworestrictionenzymesandnuc

5、leotidessequencing,theninsertedintheeukaryoticexpressionvectorpcDNA3.1.TherecombinantvectorpcDNA3.1CD44wastransfectedintoMCF7cells,andtheexpressionchangesofCD44geneandproteinweredetectedat48hourlater,respectively.Results：CD44genewasclonedfromMCF7/Adrcel

6、ls,anditseukaryoticexpressionvectorwasconstructedsuccessfully.Aftertheidentificationandsequencing,thereconstructedplasmidwasconfirmedcontainingthesequenceofCD44gene.Aftertransienttransfection,themRNAandproteinlevelofCD44genewereobviouslyupregulatedinMCF7

7、cells.ScreenedwithG418fortwoweeks,positiveclonewasobtainedfromtheabovecells.Conclusion：TheexpressionvectorpcDNA3.1CD44wasconstructedsuccessfullyandcouldbe15expressedinhumanmammarycarcinomacells.AnewCD44isoformswasfoundinMCF7/Adrcells(GeneBankNO.FJ216964)

8、；OurstudymaystartanewapproachtostudythefunctionofCD44geneandprovideamethodofgenetherapy.［Keywords］CD44；multidrugresistance；invasion；humanmammarycarcinomacellsCD44分子是一类重要的尚未归类的黏附分子,于1980年由Dalchau等［1］用单克隆抗体技术检测