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目的构建hsa-mir-520真核表达载体,并转染大肠癌细胞SW-480细胞,检测细胞中E-钙粘蛋白表达情况。方法依据miRBase数据库中hsa-mir-520序列,设计并合成寡核苷酸片段,构建hsa-mir-520表达载体和对照载体。采用脂质体转染方法分别将mir-520表达载体、及对照质粒转染大肠癌细胞SW-480细胞株。采用酶切、测序检测载体构建;Western blot检测E-钙粘蛋白的表达。结果酶切和测序表明hsa-mir-520真核表达载体构建成功。Western blot结果显示与转染对照质粒和未转染的细胞相比,转染了hsa-mir-520重组质粒的细胞中E-cadherin蛋白的表达明显增加(P<0.01),密度扫描半定量分析显示E-cadherin蛋白的表达增加了大约40%。结论成功构建了hsa-mir-520和对照的真核表达载体,hsa-mir-520质粒转染大肠癌细胞SW-480后可增加E-钙粘蛋白的表达。
Objective To construct hsa-mir-520 eukaryotic expression vector and transfect it into SW-480 cell line to detect the expression of E-cadherin. Methods According to hsa-mir-520 sequence of miRBase database, oligonucleotide fragments were designed and synthesized to construct hsa-mir-520 expression vector and control vector. The mir-520 expression vector and the control plasmid were transfected into SW-480 cell line of colorectal cancer cell by lipofectamine transfection method. The vector was constructed by restriction enzyme digestion and sequencing. The expression of E-cadherin was detected by Western blot. Results The enzyme digestion and sequencing showed that the hsa-mir-520 eukaryotic expression vector was successfully constructed. Western blot results showed that the expression of E-cadherin protein was significantly increased in cells transfected with hsa-mir-520 recombinant plasmid (P <0.01) compared with the control plasmid and untransfected cells. Semi-quantitative analysis by density scanning The expression of E-cadherin protein was shown to increase by about 40%. Conclusion The eukaryotic expression vector of hsa-mir-520 and control was constructed successfully. The expression of E-cadherin was increased after hsa-mir-520 plasmid was transfected into SW-480 cells.