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目的构建人Rac1基因shRNA表达质粒,为提高卵巢癌放化疗的敏感性奠定基础。方法根据GenBank中登录的人Rac1基因序列,应用shRNA设计软件设计并合成用于构建shRNA表达质粒的oligo DNA,构建shRNA重组质粒。将阳性Rac1基因shRNA重组质粒经脂质体介导转染卵巢癌Skov3细胞,48 h后经RT-PCR筛选有效的shRNA重组质粒。结果经限制性内切酶BamHⅠ和PstⅠ酶切鉴定出阳性Rac1基因shRNA重组质粒,序列比对分析结果与理论序列完全一致。RT-PCR检测显示,转染pGPU6/GFP/Rac1-524的Skov3细胞Rac1基因mRNA的转录水平下降。结论已成功构建人Rac1基因shRNA表达质粒,并筛选出有效的干扰质粒pGPU6/GFP/Rac1-524。
Objective To construct the shRNA expression plasmid of human Rac1 gene and lay a foundation for enhancing the sensitivity of radiotherapy and chemotherapy in ovarian cancer. Methods According to the human Rac1 gene sequence registered in GenBank, oligo DNA for constructing shRNA expression plasmid was designed and synthesized by using shRNA design software to construct shRNA recombinant plasmid. The positive Rac1 gene shRNA recombinant plasmid was transfected into ovarian cancer Skov3 cells by liposome, and after 48 h, the shRNA recombinant plasmid was screened by RT-PCR. Results Restriction endonuclease BamH Ⅰ and Pst Ⅰ digestion identified positive Rac1 gene shRNA recombinant plasmid, the sequence alignment analysis results and the theoretical sequence is exactly the same. The RT-PCR results showed that the transcription level of Rac1 mRNA in Skov3 cells transfected with pGPU6 / GFP / Rac1-524 was decreased. Conclusion The shRNA expression plasmid of human Rac1 gene has been successfully constructed and pGPU6 / GFP / Rac1-524 plasmid was screened out.