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目的:构建并鉴定靶向人转录因子STAT3基因的shRNA慢病毒载体。方法:设计合成针对人STAT3基因的shRNA序列,运用基因重组技术将其插入到含U6启动子及绿色荧光蛋白基因的慢病毒表达载体pSicoR中,构建shRNA重组质粒pSicoR-STAT3-shRNA。经双酶切及DNA测序鉴定后,利用转染试剂X-tremeGENE HP将第3代慢病毒载体共转染至HEK293细胞进行病毒包装。以阴性载体为对照,用RT-PCR和Western blot分别检测STAT3基因和蛋白表达水平。结果:双酶切及测序证实载体构建正确,在HEK293中成功包装出高滴度慢病毒颗粒,感染HEK293后STAT3基因表达和蛋白质表达水平均较对照组明显降低(P<0.05)。结论:成功构建了靶向人STAT3基因的shRNA慢病毒载体。
Objective: To construct and identify shRNA lentivirus vector targeting human STAT3 gene. Methods: The shRNA sequence of human STAT3 gene was designed and synthesized. The recombinant plasmid pSicoR-STAT3-shRNA was constructed by inserting the shRNA sequence into lentiviral expression vector pSicoR containing U6 promoter and green fluorescent protein gene by gene recombination technology. After double enzyme digestion and DNA sequencing, the third generation lentiviral vector was co-transfected into HEK293 cells with the transfection reagent X-tremeGENE HP for viral packaging. Negative vector as a control, STAT-3 gene and protein expression levels were detected by RT-PCR and Western blot. Results: The constructed vector was confirmed by double enzyme digestion and sequencing. High titer lentivirus particles were successfully packaged in HEK293 cells. The expression of STAT3 gene and protein in HEK293 cells were significantly lower than those in control group (P <0.05). Conclusion: shRNA lentivirus vector targeting human STAT3 gene was successfully constructed.