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目的构建人性别决定区Y盒6(SOX6)基因真核表达载体并观察其在KYSE30细胞内的表达。方法将人SOX6c DNA克隆到真核表达载体pc DNA3.1(+)中,经酶切和测序证实后,以Lipofectamine2000介导转染KYSE30细胞,应用RT-PCR和western blot鉴定SOX6在细胞内的表达。结果 SOX6 c DNA为2358bp的片段,重组质粒pc DNA3.1(+)-SOX6经EcoRⅠ、XhoⅠ双酶切后产生的2400bp和5400bp的片段,DNA测序证实酶切产生的2400bp片段的碱基序列与人类SOX6 c DNA完全一致。将其转染至KYSE30后,RT-PCR和western blot的结果显示SOX6能在真核细胞中正确表达。结论成功的构建了pc DNA3.1(+)-SOX6重组质粒,为后续的研究奠定基础。
Objective To construct eukaryotic expression vector of Y-box 6 (SOX6) in human sex determining region and observe its expression in KYSE30 cells. Methods Human SOX6c DNA was cloned into eukaryotic expression vector pcDNA3.1 (+). After confirmed by restriction enzyme digestion and sequencing, the recombinant human SOX6c was transfected into KYSE30 cells with Lipofectamine2000. The expression of SOX6 in cells was detected by RT-PCR and western blot expression. Results The 2358bp fragment of SOX6cDNA was obtained. The 2400bp and 5400bp fragments of the recombinant plasmid pcDNA3.1 (+) - SOX6 were digested with EcoRⅠ and XhoⅠ. The sequence of the 2400bp fragment was confirmed by DNA sequencing. Human SOX6 c DNA is identical. After transfection to KYSE30, the results of RT-PCR and western blot showed that SOX6 can be correctly expressed in eukaryotic cells. Conclusion The pcDNA3.1 (+) - SOX6 recombinant plasmid was successfully constructed, which laid the foundation for further research.