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1、干擾AFP蛋白表達(dá)shRNA表達(dá)載體構(gòu)建及活性鑒定作者：張國英劉明社趙中夫*張蕓楊慧武延雋【摘要】目的：利用含報(bào)告基因EGFP的pGenes訂-1質(zhì)粒構(gòu)建干擾AFP蛋白表達(dá)的短發(fā)夾RNA(shorthairpinRNA,shRNA)表達(dá)的載體，并進(jìn)行活性鑒定。方法：設(shè)計(jì)表達(dá)短發(fā)夾RNA的互補(bǔ)DNA序列，經(jīng)退火成雙鏈，克隆至帶有U6啟動(dòng)子的質(zhì)粒載體pGenes訂T中，構(gòu)建重組質(zhì)粒，轉(zhuǎn)化大腸桿菌DH5a菌株，擴(kuò)增，提取質(zhì)粒，酶切鑒定后測序分析。構(gòu)建正確的重組質(zhì)粒經(jīng)脂質(zhì)體轉(zhuǎn)染HepG2細(xì)胞，檢測轉(zhuǎn)染率和培養(yǎng)細(xì)胞上清的AFP含量變化，確定重組質(zhì)粒的活性。結(jié)果：構(gòu)建了靶向AFP蛋白的2個(gè)shRN
2、A重組質(zhì)粒載體pGenesil-1-siAFPl和pGenesil-l-siAFP2o酶切鑒定和測序分析，shRNA編碼序列與設(shè)計(jì)的片段完全一致，經(jīng)酶切凝膠電泳證實(shí)載體構(gòu)建成功。重組質(zhì)粒有效轉(zhuǎn)染HepG2細(xì)胞并顯著抑制AFP表達(dá)。結(jié)論：成功構(gòu)建具有活性的、能夠表達(dá)shRNA靶向干擾AFP蛋白的2個(gè)重組質(zhì)粒載體?！娟P(guān)鍵詞】AFP蛋白R(shí)NAishRNA重組質(zhì)粒載體AbstractObjective：ToconstructtherecombinantplasmidexpressingtwoAFP—targetedshorthairpinRNA(shRNA)withpGenesil-1plas
3、midsvector,andDetecttheActivityofRecombinantPlasmidinHepG2cells.Methods：TwopairsofDNAsequencesweredesignedandsynthesized,andformedcomplementarychainsbyannealingrespectively.Thentheobtainedproducts,siAFPlandsiAFP2,wereinsertedintoplasmidvectorpGenesil-1withU6promoter.Therecombinantplasmidsweretra
4、nsformingintotheEscherichiacolistrainDH5aforscreeningandamplifying.ThesequeneeanalysisoftheplasmidsandidentifiedbySalldigestionwascarriedout?ThebiologicalactivityofrecombinantplasmidwasdetectedbytransfectingintotheHepG2cellline?Results:ThetwoAFP-targetedshRNAsexpressingframeweresuccessfullyinser
5、tedintothepGensil-1plasmidvectorrespectively,andtheobtainedshRNAscodingsequenceswereconsistentwiththedesignedfragments.TherecombinantplasmidinhibitedeffectivelytheexpressionofAFPgeneinHepG2cells.Conclusion：TheconstruetionoftworecombinantplasmidsofAFP-targetedshRNAwassuccessful.Tworecombinantplas
6、midsexpressingAFP—targetedshRNAinhibitedeffectivelytheexpressionofAFPgeneinHepG2cells.KeyWordsAFP；RNAinterference；ShorthairpinRNA；HepG2cells長期以來，人們只是把AFP作為一個(gè)腫瘤標(biāo)志物，用于原發(fā)性肝癌(HCC)的診斷指標(biāo)之一。諸多研究關(guān)注于檢測甲胎蛋白(alpha-fetoprotein,AFP)對原發(fā)性肝癌診斷運(yùn)用，而對AFP和腫瘤細(xì)胞發(fā)生及增殖的關(guān)系并不完全清楚［1?3］o近年發(fā)展起來的RNA干擾(RNAinterference,RNAi)技術(shù)
7、為探索AFP的生物學(xué)功能提供了有力的工具。RNAi技術(shù)是通過導(dǎo)入細(xì)胞19nt?23nt的小分子干擾RNA(smallinterferenceRNA,siRNA),降解同源mRNA,高效、特異性阻斷目的基因表達(dá)［4?7］。本研究針對人AFP基因設(shè)計(jì)了2個(gè)位點(diǎn)的shRNA表達(dá)序列，用人U6啟動(dòng)子和RNApolymeraselll(PolIII)終止信號，構(gòu)建針對AFP的特異性shRNA真核表達(dá)載體，并轉(zhuǎn)染HepG2細(xì)胞鑒定活性，探索RNAi技術(shù)在沉
