CAJ | 학술논문

目的构建MEK2-siRNA表达质粒,转染胶质瘤U87细胞并研究其对内源性MEK2表达的敲减效果.方法化学合成4条60个碱基并能转录siRNA发卡结构的DNA寡核苷酸,退火形成2条双链DNA,以T4连接酶接入BglⅡ和HindⅢ双酶切后的pSUPER.basic载体中.EcoRⅠ和HindⅢ双酶切鉴定出阳性重组克隆,抽提质粒并转染胶质瘤U87细胞,Western blotting检测MEK2蛋白的表达水平.结果酶切鉴定和测序结果表明MEK2-siRNA质粒构建成功,Western blotting结果筛选出较好的MEK2-siRNA质粒.未转染阴性对照质粒MEK2表达水平为0.105±0.023,转染筛选质粒后表达水平为0.030±0.006.结论成功构建了MEK2-siRNA真核表达载体,并证明其能下调胶质瘤U87细胞中MEK2蛋白的表达水平,为RNA干扰技术应用于胶质瘤的基因治疗提供了一定的实验依据.
목적 구건MEK2-siRNA표체질립,전염효질류U87세포병연구기대내원성MEK2표체적고감효과.방법 화학합성4조60개감기병능전록siRNA발잡결구적DNA과핵감산,퇴화형성2조쌍련DNA,이T4련접매접입BglⅡ화HindⅢ쌍매절후적pSUPER.basic재체중.EcoRⅠ화HindⅢ쌍매절감정출양성중조극륭,추제질립병전염효질류U87세포,Western blotting검측MEK2단백적표체수평.결과 매절감정화측서결과표명MEK2-siRNA질립구건성공,Western blotting결과사선출교호적MEK2-siRNA질립.미전염음성대조질립MEK2표체수평위0.105±0.023,전염사선질립후표체수평위0.030±0.006.결론 성공구건료MEK2-siRNA진핵표체재체,병증명기능하조효질류U87세포중MEK2단백적표체수평,위RNA간우기술응용우효질류적기인치료제공료일정적실험의거.
Objective To construct a eukaryotic expression vector for MEK2-siRNA to explore the expression of this endogenous MEK2 in glioma U87 cell line. Methods Four single-stranded template DNAs encoding siRNA against MEK2, each consisting of 60 bp, were synthesized chemically;based on these 4 single-stranded template DNAs, 2 double-stranded DNAs were formed by annealing,then identified by restriction analysis and inserted into vector pSUPER.basic by T4 ligase. Positive recombinants were indentified by EcoRI and HindⅢ double digestion and transfected into the U87 cells.The protein expression level of MEK2 was determined by using Western blotting. Results Both restriction analysis and sequencing proved that the eukaryotic expression vector for MEK2-siRNA was constructed correctly. MEK2-siRNA plasmid screened out by Western blotting. The MEK2 expression level in negative control group was (0.105±0.023) and that in transfected group was (0.030±0.006).Conclusion The eukaryotic expression vector for MEK2-siRNA is successfully constructed, which down-regulates the transcription of MEK2 protein in U87 cells. It provides a certain experimental basis for gene therapy of glioma by RNAi technique.