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《靶向表皮生長因子受體shRNA表達(dá)載體的構(gòu)建和鑒定》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在應(yīng)用文檔-天天文庫。
1、靶向表皮生長因子受體shRNA表達(dá)載體的構(gòu)建和鑒定【摘要】目的構(gòu)建靶向表皮生長因子受體(EGFR)的短發(fā)卡狀RNA(shRNA)質(zhì)粒表達(dá)載體并進(jìn)行鑒定。方法①根據(jù)人類EGFR的mRNA序列，設(shè)計(jì)4條干擾片段，以PGPU6/GFP/Neo質(zhì)粒為載體，構(gòu)建shRNA重組體，轉(zhuǎn)化入DH5a大腸桿菌，提取質(zhì)粒，進(jìn)行酶切和測序鑒定;②采用脂質(zhì)體Lipofectamin2000分別轉(zhuǎn)染人皮膚鱗狀細(xì)胞癌細(xì)胞株Colo-16細(xì)胞，用G418篩選陽性克隆。結(jié)果①經(jīng)酶切及測序鑒定，成功構(gòu)建了針對EGFR的4個(gè)重組干擾質(zhì)粒;②成功轉(zhuǎn)染Colo-16細(xì)胞，經(jīng)篩選后得到穩(wěn)定表達(dá)的細(xì)胞克隆。結(jié)論利用
2、RNAi技術(shù)原理成功構(gòu)建針對EGFR的shRNA重組質(zhì)粒，并轉(zhuǎn)染入Colo-16細(xì)胞?！娟P(guān)鍵詞】表皮生長因子受體;RNA干擾;短發(fā)卡狀RNA;轉(zhuǎn)染 Abstract：ObjectiveToestablishandidentifytheplasmidexpressionvectorencodingshorthairpinRNA(shRNA)targetingepidermalgrowthfactorreceptor(EGFR)ofhumangene.Methods①FourshorthairpinRNAs(shRNA)weredesignedaccordingtotheh
3、omosapiensEGFRmRNAID,andthenrecombinantshRNAwasreconstructed,withPGPU6/GFP/Neoplasmidsasvectors,respectively.TheplasmidsweretransferredintoE.coliDH5αforplasmidextractionandthesubsequentidentificationby12enzymedigestingandsequencing.②FourrecombinantplasmidsweretransfectedintoColo-16cellswi
4、thlipofectamine2000andwerescreenedforpositiveclonesbyG418.Results①Theresultsofenzyme-digestionsequencingconfirmedthatfourplasmidscontainingshRNAweresuccessfullyconstructed.②FourrecombinantplasmidsweresuccessfullytransfectedintoColo-16cellsandthecellcloneswithstableexpressionwereobtained.C
5、onclusionWithRNAitechnology,therecombinantshRNAplasmidstargetingEGFRweresuccessfullyestablishedandtransfectedintoColo-16cells. Keywords:epidermalgrowthfactorreceptor;RNAinterference;smallhairpinRNA;transfection 表皮生長因子受體(epidermalgrowthfactorreceptor,EGFR)屬于表皮生長因子家族[1](erbB家族)，為原癌基因C-erb
6、B(HER-1)的表達(dá)產(chǎn)物，是一種細(xì)胞膜表面的糖蛋白受體，它與表皮生長因子(epidermalgrowthfactor,EGF)、轉(zhuǎn)化生長因子(transferringgrowthfactor12,TGF)等配體結(jié)合，能激活酪氨酸激酶受體，導(dǎo)致酪氨酸殘基的磷酸化，促進(jìn)細(xì)胞無節(jié)制地增殖分裂，導(dǎo)致惡性腫瘤的發(fā)生[2]。阻斷EGFR可抑制腫瘤生長，其作用機(jī)制可能與抑制細(xì)胞周期進(jìn)程、加速細(xì)胞凋亡、抑制血管生成、抑制浸潤和轉(zhuǎn)移、增強(qiáng)化、放療敏感性等有關(guān)[3]。小干擾RNA(siRNA)[4]與同源性mRNA結(jié)合,可靶向切斷降解此mRNA,在蛋白翻譯前抑制基因表達(dá)[5]。我們構(gòu)建編碼
7、EGFR的短發(fā)卡狀RNA(shRNA)質(zhì)粒表達(dá)載體,并對其進(jìn)行鑒定,為皮膚鱗癌的靶向治療尋找新的途徑?！ ?材料和方法 1.1材料空質(zhì)粒PGPU6/GFP/Neo為Genepharma公司產(chǎn)品。各種限制性內(nèi)切酶、T4DNA連接酶為美國Promega公司產(chǎn)品。Lipofectamine2000轉(zhuǎn)染試劑盒為Invitrogen公司產(chǎn)品。感受態(tài)菌DH5a購于博大泰克公司;質(zhì)粒抽提試劑盒購于Promega公司;G418、卡那霉素購于Sigma公司;人皮膚鱗狀細(xì)胞癌細(xì)胞株Colo-16細(xì)胞由中國醫(yī)學(xué)科學(xué)院皮膚病研究所周武慶教授惠贈(zèng);R
