论文部分内容阅读
目的构建带FLAG标签的细胞S期激酶相关蛋白2(Skp2)基因真核表达载体pc DNA3-FLAG-Skp2并检测其对MCF-7乳腺癌细胞生长及细胞周期的影响。方法采用反转录PCR从MDA-MB-231乳腺癌细胞中扩增Skp2基因,构建重组真核表达载体pc DNA3-FLAG-Skp2。酶切及测序鉴定重组质粒正确后,脂质体介导转染至MCF-7乳腺癌细胞中。采用Western blot法检测FLAG-Skp2的表达,Alamar blue比色法绘制细胞生长曲线,流式细胞术检测细胞周期。结果 PCR扩增片段与预期片段大小相符,测序结果与Gen Bank结果一致。Western blot结果显示质粒转染48 h后,细胞成功表达融合蛋白FLAG-Skp2。过表达Skp2的MCF-7乳腺癌细胞较对照组生长速度明显加快,S期细胞显著增多。结论成功构建人Skp2真核表达载体pc DNA3-FLAG-Skp2。过表达Skp2的MCF-7细胞生长加快,S期细胞增多。
Objective To construct the eukaryotic expression vector pcDNA3-FLAG-Skp2 with FLAG-tagged S-phase kinase-related protein 2 (Skp2) gene and study its effect on the growth and cell cycle of MCF-7 breast cancer cells. Methods Skp2 gene was amplified from MDA-MB-231 breast cancer cells by reverse transcription polymerase chain reaction (RT-PCR) and the recombinant eukaryotic expression vector pcDNA3-FLAG-Skp2 was constructed. Restriction enzyme digestion and DNA sequencing confirmed that the recombinant plasmid was correctly transfected into MCF-7 breast cancer cells by liposome. The expression of FLAG-Skp2 was detected by Western blot, the cell growth curve was drawn by Alamar blue colorimetric assay, and the cell cycle was detected by flow cytometry. Results The PCR amplified fragment was consistent with the expected fragment size, and the sequencing result was consistent with that of Gen Bank. Western blot results showed that the fusion protein FLAG-Skp2 was successfully expressed 48 h after transfection. The growth rate of MCF-7 breast cancer cells overexpressing Skp2 was significantly higher than that of the control group, and the number of S phase cells was significantly increased. Conclusion The human Skp2 eukaryotic expression vector pcDNA3-FLAG-Skp2 was successfully constructed. MCF-7 cells overexpressing Skp2 grew faster and S phase increased.