【摘要】 目的构建人notch1-shRNA慢病毒表达载体,为Notch信号通路的相关研究奠定基础。方法根据人notch1基因mRNA序列设计并合成两条互补的DNA单链寡核苷酸,将退火后形成的双链连接于pENTR/U6质粒,通过与pLenti6/BLOCK-iT-DEST载体的LR重组,构建notch1-shRNA慢病毒表达载体,经脂质体介导入293FT细胞,包装成慢病毒。用该慢病毒感染人神经母细胞瘤系SH-SY5Y细胞,RT-PCR和Westernblot法检测细胞notch1基因的表达。结果目的序列成功连接于载体并包装成较高滴度的慢病毒,细胞mRNA和蛋白质的检测均表明构建的notch1-shRNA慢病毒表达载体LR-ND-A能显著抑制SH-SY5Y细胞notch1基因的表达。结论成功构建人notch1-shRNA慢病毒表达载体。更多还原

【Abstract】 Objective To construct a lentiviral vector expressing shRNA targeting human notch1 gene for the investigation of Notch signal pathway.Methods complementary single-stranded DNA oligos were designed and synthesized according to the mRNA sequence of human notch1.The single-standed oligos were annealed to generate ds-oligos and then linked with linearized pENTR/U6 to obtain entry clones.An LR recombination reaction was performed between the pENTR/U6 entry constructs and pLenti6/BLOCK-iT-DEST to generate lentiviral vectors expressing small hairpin RNA(shRNA)targeting notch1.The recombinant lentivirus was generated by cotransfection of 293FT cells with pLenti6/BLOCK-iT constructs and viral packaging mix.Human neuroblastoma SH-SY5Y cells were infected with the recombinant lentivirus expressing notch1-shRNA and the expression level of notch1 in cells were examined by RT-PCR and Western blotting analysis.Results The recombinant letiviral vector expressing notch1-shRNA was successfully constructed and high liter recombinant lentivirus were generated.The expression of notch1 mRNA and protein were suppressed in cells infected with the lentivirus LR-ND-A expressing shRNA of notch1.Conclusion Lentiviral vectors expressing shRNA targeting human notch1 gene were successfully constructed.更多还原