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《乙酰肝素酶基因靶向特異性rna干擾重組表達(dá)載體的構(gòu)建及》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在工程資料-天天文庫(kù)。
1、窯2340窯南方醫(yī)科大學(xué)學(xué)報(bào)(JSouthMedUniv)2010;30(10)乙酰肝素酶基因靶向特異性RNA干擾重組表達(dá)載體的構(gòu)建及篩選劉陽(yáng)袁霍強(qiáng)袁許立薇袁蔣志文淵蚌埠醫(yī)學(xué)院藥學(xué)系袁安徽省生化藥物工程技術(shù)研究中心袁安徽蚌埠233030冤摘要院目的構(gòu)建對(duì)人乙酰肝素酶淵HPA冤基因有特異性抑制作用的短發(fā)夾狀RNA表達(dá)載體遙方法根據(jù)GenBank數(shù)據(jù)庫(kù)提供的人HPA基因核苷酸序列袁按照shRNA設(shè)計(jì)原則設(shè)計(jì)5對(duì)特異性的寡核苷酸序列及陰性對(duì)照袁分別克隆到pGPU6/GFP/Neo載體中袁酶切鑒定和測(cè)序驗(yàn)證陽(yáng)性克隆遙用脂質(zhì)體轉(zhuǎn)染入MDA-MB-
2、231細(xì)胞袁分別以熒光定量PCR堯Westernblot分析其對(duì)HPA在mRNA水平和蛋白質(zhì)水平上表達(dá)的影響遙結(jié)果經(jīng)酶切堯測(cè)序鑒定均證實(shí)重組質(zhì)粒構(gòu)建成功遙在構(gòu)建的5個(gè)載體中袁HPSE-1組及HPSE-5組中MDA-MB-231細(xì)胞HPA基因mRNA和蛋白質(zhì)表達(dá)水平明顯下降袁而對(duì)照組未發(fā)生明顯改變遙結(jié)論成功構(gòu)建了2個(gè)靶向人HPA的shRNA表達(dá)載體袁轉(zhuǎn)染MDA-MB-231細(xì)胞后可高效抑制HPA基因表達(dá)袁為進(jìn)一步研究HPA的表達(dá)與乳腺癌遷移和侵襲的機(jī)制奠定了基礎(chǔ)遙關(guān)鍵詞院乙酰肝素酶曰RNA干擾曰MDA-MB-231細(xì)胞中圖分類號(hào)院R915
3、文獻(xiàn)標(biāo)志碼院A文章編號(hào)院1673-4254(2010)10-2340-03ConstructionandscreeningofspecificshorthairpinRNAvectortargetingheparanasegeneLIUYang,HUOQiang,XULi-wei,JIANGZhi-wenSchoolofPharmacy,BengbuMedicalCollege,AnhuiEngineeringTechniqueResearchCenterforBiochemicalDrugs,Bengbu233030,ChinaAbs
4、tract:ObjectiveToconstructshorthairpinRNA(shRNA)expressionvectorsofRNAforspecificsilencingofheparanase(HPA)gene.MethodsThegenomicsequenceofHPAgenewasretrievedfromGenBankandthecDNAencodingshRNAforHPAgenesilencingwasdesigned.Fivespecificinterferencesequencesandarandomnegat
5、ivecontrolsequencewereinsertedintothevectorpGPU6/GFP/Neo.Afterverificationbyrestrictionenzymedigestionandsequenceanalysis,therecombinantvectorsweretransfectedintoMDA-MB-231cellsvialipofectamin.FluorescentquantitativePCRandWesternblottingwereemployedtodetecttheexpressiono
6、fHPAgeneexpressionsinthetransfectedcellsatthemRNAandproteinlevels,respectively.ResultsBothrestrictionanalysisandsequencingconfirmedcorrectconstructionoftheshRNAvectors.TransfectedwiththespecificsiRNAvectorsHPSE-1andHPSE-5resultedinsignificantlydecreasedexpressionlevelofH
7、PAproteininMDA-MB-231cells,whilenegativecontrolvectorproducednosignificantchangesinHPAexpressions.ConclusionWehaveobtainedtwoshRNAvectorswhichcansignificantlydown-regulateHPAexpressionsinMDA-MB-231cells,whichfacilitatesfurtherinvestigationoftheroleHPAmayplayintheinvasive
8、nessandmetastasisofhumanbreastcancer.Keywords:heparanase;RNAinterference;MDA-MB-231cells腫瘤的發(fā)生和發(fā)展與細(xì)胞外基質(zhì)和
