DNA-PKcs短发夹RNA载体的构建及其表达

The Construction of DNA-Pkcs Short-hairpin siRNA Recombinant Vector and Its Expression in Vitro

摘要: 目的构建DNA-PKcsshRNA表达载体,观察该基因的抑制对A2780细胞增殖活性的影响。方法将针对人DNA-PKcs基因的不同部位设计的shRNA插入到真核表达质粒并转染人卵巢A2780细胞,RT-PCR和WesterBlot检测其对该基因mRNA和蛋白水平的抑制效率,MTT法测定DNA-PKcs基因的抑制对肿瘤细胞增殖活性的影响。结果 DNA测序分析证实DNA-PKcs shRNA表达载体pSIRENDNA-PKcs shRNA构建成功并在细胞中表达,转染重组载体的A2780细胞DNA-PKcs的表达在mRNA和蛋白水平均明显下降;重组载体转染细胞增殖活性明显降低。结论成功构建DNA-PKcs shRNA表达载体为进一步研究DNA损伤修复基因DNA-PKcs奠定了基础;DNA-PKcs表达的抑制可能会导致肿瘤细胞增殖活性降低。

Abstract: Objective To construct eukaryotic vector expressing shRNA(short hairpin RNA) of DNA-PKcs and study the significance of superexpression of DNA-PKcs in A2780 cells. Methods Designing two different shRNA targeting the coding sequence of DNA-PKcs, the pSIREN-DNA-PKcs shRNA was constructed by inserting the designed shRNA to the plasmid vector pSIREN.The human ovarian cancer cell strain A2780 was transfected by pSIREN-DNA-PKcs shRNA. The Expression of DNA-Pkcs in transfercted cells was detected by RT-PCR and West...