人cd44基因真核表達(dá)載體構(gòu)建及在乳腺癌細(xì)胞中的表達(dá)

《人cd44基因真核表達(dá)載體構(gòu)建及在乳腺癌細(xì)胞中的表達(dá)》由會員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在學(xué)術(shù)論文-天天文庫。

1、人CD44基因真核表達(dá)載體構(gòu)建及在乳腺癌細(xì)胞中的表達(dá)作者：房新建許文林張徐錢暉陳琛方莉莉陳巧云【摘要】目的：從人類乳腺癌耐多柔比星細(xì)胞株細(xì)胞（MCF7/Adr)中克隆CD44基因并構(gòu)建CD44基因真核表達(dá)載體pcDNA3.1CD44;將pcNDA3.1CD44穩(wěn)定轉(zhuǎn)染入人乳腺癌細(xì)胞株（MCF7）細(xì)胞中。方法：從MCF7/Adr細(xì)胞中提取總RNA進(jìn)行RTPCR，獲得全長的cDNA模板；將含有CD44基因的cDNA模板使用限制性內(nèi)切酶進(jìn)行雙酶切獲得帶有酶切位點(diǎn)的CD44基因,再將帶有酶切位點(diǎn)的CD4

2、4基因經(jīng)TA克隆后測序驗(yàn)證，然后亞克隆入真核表達(dá)載體pcDNA3.1中，雙酶切鑒定后再次測序驗(yàn)證。脂質(zhì)體法將pcDNA3.1CD44質(zhì)粒轉(zhuǎn)染到MCF7細(xì)胞中，48小時后RTPCR和流式細(xì)胞術(shù)檢測轉(zhuǎn)染前后CD44基因、蛋白在MCF7細(xì)胞的表達(dá)變化。結(jié)果：成功從人MCF7/Adr細(xì)胞中克隆獲得CD44基因并構(gòu)建真核表達(dá)載體，轉(zhuǎn)染后在MCF7中檢測到CD44基因mRNA和蛋白水平表達(dá)上調(diào)；將上述表達(dá)CD44基因及蛋白的瞬時轉(zhuǎn)染MCF7細(xì)胞經(jīng)G418篩選兩周后獲得陽性克隆。結(jié)論：成功克隆和建立人CD4

3、4基因真核表達(dá)質(zhì)粒；pcDNA3.1CD44能在MCF7細(xì)胞中表達(dá)；在MCF7/Adr中新發(fā)現(xiàn)一種CD44基因的變異體(基因庫號FJ216964)；這為進(jìn)一步研究其基因功能打下了基礎(chǔ)。【關(guān)鍵詞】CD44；多藥耐藥；侵襲；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分子是一類重要的尚未歸類的黏附分子,于1980年由Dalchau等［1］用單克隆抗體技術(shù)檢測