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1、1α真核表達(dá)質(zhì)粒的構(gòu)建及其在MCF-7細(xì)胞中的表達(dá)2006年8月第26卷第8期基礎(chǔ)醫(yī)學(xué)與臨床Basic&ClinicalMedicineAugust2006V01.26No.8文章編號(hào):1O01-6325(2006)08-0888-06研究論文pcDNA3一HIF一1O/.真核表達(dá)質(zhì)粒的構(gòu)建及其在MCF一7細(xì)胞中的表達(dá)蘇燕,李宏偉,張靜,修瑞娟(中國(guó)醫(yī)學(xué)科學(xué)院中國(guó)協(xié)和醫(yī)科大學(xué)微循環(huán)研究所,北京100005)摘要:目的構(gòu)建低氧誘導(dǎo)因子let(HIF.1et)真核表達(dá)質(zhì)粒,并在MCF-7細(xì)胞中進(jìn)行功能驗(yàn)證.方法
2、利用RT.PCR擴(kuò)增帶有XbaI和HindIll酶切位點(diǎn)的HIF-let編碼基因,插入T載體測(cè)序正確后,克隆人pcDNA3真核表達(dá)載體,然后將其轉(zhuǎn)染MCF-7細(xì)胞.Westernblot和雙熒光素酶報(bào)告基因法分別檢測(cè)HIF-1et表達(dá)及其DNA結(jié)合活性;實(shí)時(shí)定量PCR檢測(cè)HIF-let對(duì)低氧誘導(dǎo)基因C—METmRNA表達(dá)的影響;Caspase3/7活性檢測(cè)來初步判斷HIF-1et對(duì)低氧MCF-7細(xì)胞凋亡的影響.結(jié)果酶切及測(cè)序結(jié)果證明~DNA3一HIF.1et表達(dá)質(zhì)粒的DNA序列完全正確.將此質(zhì)粒轉(zhuǎn)染MCF-7細(xì)胞后
3、,HIF-1et蛋白表達(dá)明顯增加,它不僅增強(qiáng)了pGL3-EPO-HRE報(bào)告基因活性而且促進(jìn)rC—METmRNA的表達(dá),同時(shí)還降低了低氧MCF-7細(xì)胞中Caspase3/7的水平.結(jié)論pcDNA3-HIF-let真核表達(dá)質(zhì)粒構(gòu)建成功,它不但具有很強(qiáng)的DNA結(jié)合和誘導(dǎo)活性,而且在細(xì)胞低氧過程中具有一定的抗凋亡作用.關(guān)鍵詞:低氧;低氧誘導(dǎo)因子.1ot;質(zhì)粒構(gòu)建;基因表達(dá);凋亡中圖分類號(hào):R363文獻(xiàn)標(biāo)識(shí)碼:AConstructionofpcDNA3—-HIF—-loteukaryoticexpressionvectora
4、nditsexpressioninMCF一7cellsSUYan,LIHong—wei,ZHANGJing,XIURui-juan(InstituteofMierocirculation,PekingUnionMedicalCollege&ChineseAcademyofMedicalSciences,Beijing100005,China)Abstract:ObjectiveToconstructpcDNA3一HIF一1僅eukaryoticexpressionvectorandtoinvestigate
5、itsfunctioninbreastcancerMCF-7cells.MethodsRT—PCRwasappliedtoamplifyhumanHIF一1僅cDNAfromMCF-7cellswithapairofsequencespecificprime~carryingarestrictionenzymesiteXbaIorHind11Ioneach5end.HIF一1僅cDNAwasinsertedintopMD18一Taftersequencing,andtheninsertedintopcDNA3vec
6、tor.TherecombinantvectorwastransfectedintoMCF-7cellstoobserveitsexpressionandfunctionbyWesternblotanddual—luciferasereportergeneassay.Realtime—PCRwasperformedtodetecttheinducibleexpressionofC—METmRNAbyHIF一1僅.theanti—apoptoticeffectofHIF一1underhypoxiawasanalyse
7、dbydetectingtheCaspase3/7activity.ResultsThesequenceofpcDNA3一HIF一1僅isidenticalwiththegenebank.ItenhancestheexpressionofHREreportergeneandC—METmRNA.butdecreasestheCaspase3/7activityinMCF-7cellsunderhypoxia.ConclusionThepcDNA3一HIF一1僅eukaryoticexpressionvectorwas
8、successfullyconstructed,itnotonlyhasstrongDNAbindingandinducingactivity,butalsohasanti—apoptoticeffectinhypoxiaMCF一7cells.Keywords:hypoxia;hypoxiainduciblefactor—let;plasmidconstru
