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目的:构建p70核糖体蛋白S6激酶1(p70 ribosomal protein S6 kinase 1,p70 S6K1)及p85 S6K1基因的真核表达载体pcDNA3.1(-)-flag-p70 S6K1和pcDNA3.1(-)-flag-p85 S6K1,并鉴定其在人乳腺癌MCF-7细胞内的表达及功能。方法:以pRK7-HA-S6K1为模板,采用PCR扩增出目的基因片段p70 S6K1、p85 S6K1,克隆入真核表达载体pcDNA3.1(-)-flag构建重组表达载体pcDNA3.1(-)-flag-p70 S6K1和pcDNA3.1(-)-flag-p85 S6K1,采用PCR、双酶切和DNA测序鉴定。将重组载体转染MCF-7细胞,24 h后采用Western blotting方法检测细胞内p70 S6K1、p85 S6K1蛋白的表达;同时向转染细胞内加入1 mmol/L H2O2处理36 h,观察p70 S6K1、p85 S6K1蛋白对H2O2诱导的细胞死亡的影响。结果:成功扩增得到p70 S6K1、p85 S6K1基因片段并构建重组真核表达载体pcDNA3.1(-)-flag-p70 S6K1和pcDNA3.1(-)-flag-p85 S6K1,重组载体经PCR、双酶切鉴定均出现p70 S6K1和p85S6K1预期条带,DNA测序结果显示其全长基因阅读框完整、正确。重组载体在MCF-7细胞中高效表达flag-p70 S6K1和flag-p85S6K1,且p85 S6K1能增强H2O2诱导的细胞死亡。结论:成功构建重组真核表达载体pcDNA3.1(-)-flag-p70 S6K1和pcDNA3.1(-)-flag-p85 S6K1,均能在MCF-7细胞中高效表达,且p85 S6K1能够增强H2O2诱导的细胞死亡。
OBJECTIVE: To construct eukaryotic expression vectors pcDNA3.1 (-) - flag-p70 S6K1 and pcDNA3.1 (-) - flag of p70 ribosomal protein S6 kinase 1 (p70 S6K1) and p85 S6K1 -p85 S6K1, and to identify its expression and function in human breast cancer MCF-7 cells. Methods: The p70 S6K1 and p85 S6K1 genes were amplified by PCR using pRK7-HA-S6K1 as a template and cloned into the eukaryotic expression vector pcDNA3.1 (-) - flag to construct the recombinant expression vector pcDNA3.1 (-) - flag-p70 S6K1 and pcDNA3.1 (-) - flag-p85 S6K1 were identified by PCR, double enzyme digestion and DNA sequencing. The expression of p70 S6K1 and p85 S6K1 was detected by Western blotting 24 h after MCF-7 cells were transfected with recombinant vector. The cells were treated with 1 mmol / L H2O2 for 36 h, and the expression of p70 S6K1 and p85 Effect of S6K1 protein on H2O2 induced cell death. Results: The p70 S6K1 and p85 S6K1 gene fragments were successfully amplified and the recombinant eukaryotic expression vectors pcDNA3.1 (-) - flag-p70 S6K1 and pcDNA3.1 (-) - flag-p85 S6K1 were constructed. Enzyme digestion showed p70 S6K1 and p85S6K1 expected bands, DNA sequencing results showed that the full-length gene reading frame is complete and correct. The recombinant vector highly expressed flag-p70 S6K1 and flag-p85S6K1 in MCF-7 cells, and p85 S6K1 enhanced H2O2-induced cell death. CONCLUSION: The recombinant eukaryotic expression vector pcDNA3.1 (-) - flag-p70 S6K1 and pcDNA3.1 (-) - flag-p85 S6K1 can both be efficiently expressed in MCF-7 cells and p85 S6K1 can enhance the activity of H2O2 Induced cell death.