prmt2基因的mirna载体构建及其活性鉴定word论文.docxVIP

PRMT2基因的miRNA载体构建及其活性鉴定硕士研究生：唐焱导师：姜浩教授中文摘要目的：构建人蛋白质精氨酸甲基转移酶2（protein argininemethyltransferase2, PRMT2）基因的miRNA真核表达载体，利用microRNA靶向PRMT2基因，鉴定其转染乳腺癌细胞株MCF7 后的生物活性。方法：根据PRMT2基因序列设计合成4对pre-miRNA片段，定向克隆到pcDNATM6.2-GW/EmGFP-miR真核表达载体上，采用菌落PCR和测序分析鉴定插入序列的完整性；并将重组载体转染至MCF7 细胞株中，采用Westernblot方法鉴定重组载体转染MCF7 细胞后对PRMT2 蛋白表达的干扰效果，以及萤火虫荧光素酶双报告基因系统检测PRMT2 及其重组体对ERα转录活性影响。结果：1．构建的重组体插入片段的碱基序列完全正确，间接免疫荧光法和western blot 法检测表明：重组体稳定转染MCF7 细胞后可成功干扰PRMT2 基因的表达。2．重组体转染组miPRMT2-1，miPRMT2-2，miPRMT2-3，miPRMT2-4四个重组体转染组的蛋白表达水平中，miPRMT2-3 重组转染体蛋白表达减低最明显。3.与未转染组及control-miRNA组相比, 稳定转染miPRMT2的MCF7细胞对雌激素的敏感性明显下降。4. PRMT2及其重组体对ERE-LUC转录活性的抑制是雌激素依赖的。在无E2刺激的条件下，PRMT2及其重组体对ERE-LUC转录活性无明显影响。结论：21．成功构建了靶向PRMT2 基因的microRNA真核表达载体，而且重组体能够抑制PRMT2 蛋白表达。2．靶向PRMT2 基因的microRNA重组体可以抑制ERα介导的ERE-LUC 的转录活性。关键词：乳腺癌；微小RNA类；蛋白质精氨酸甲基转移酶23ConstructionandactivityidentificationofmiRNAeukaryoticexpressingvectorofPRMT2AbstractObjective:To construct PRMT2 gene expressingeukaryoticmiRNArecombiants，and to identify biological activityof recombinat in breast cancercell lineMCF7 cells aftertransfection.Methods:Accordingto sequenceof PRMT2 gene, thepre-miRNAwas designedand synthesized, then cloned into theGFPreporter pcDNATM6.2-GW/EmGFP-miR, and transfected into MCF7 cell line.Theintegrityof inserted fragment sequencewas tested through colonyPCR and sequence analysis , and the biologicalactivityof recombinants was identified bydetecting the interference efficiencyofPMRT2 employingWestern blotting, and thesystem offireflyluciferasereportergenewas used to detect theinfluenceof thePRMT2 and recombinants on the transcription of ER α.Results:Sequences of insert fragment in fourmiRNAexpressingrecombinants was proved to be correct, and the expression ofPRMT2 was inhibited after the recombinants transient transfection in MCF7 cells.Duringthe four miRNAexpressingrecombinants groupsuchas miPRMT2-1 miPRMT2-2 miPRMT2-3 and miPRMT2-4, the expression ofprotein reducedobvious the most in thegroupofmiPRMT2-3.Comparewith theuntransfectedgroup and