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目的构建连环蛋白p120特异性shRNA重组慢病毒载体,感染胰腺癌PANC-1细胞,鉴定干扰效果。方法针对p120ctn mRNA设计合成3对shRNA序列,连接入酶切线性化的慢病毒载体pGCSIL-GFP,PCR鉴定及测序正确后与构建成功的含p120ctn基因的载体pGC-FU-p120ctn-3FLAG共转染293T细胞,蛋白质印迹分析筛选有效shRNA序列,包装慢病毒,测定病毒滴度。重组慢病毒感染胰腺癌PANC-1细胞,实时定量PCR及蛋白质印迹分析鉴定干扰效果。结果 PCR及测序证实成功构建出p120ctn-shRNA-LV慢病毒载体,病毒滴度达到3×109TU/ml。重组慢病毒感染PANC-1细胞后,实时定量PCR提示PANC-1细胞p120ctn mRNA表达下降82.6%(P<0.05),蛋白质印迹分析提示p120ctn蛋白表达较转染前显著下降。结论成功构建出p120ctn基因shRNA慢病毒载体,为进一步研究p120ctn在胰腺癌浸润及转移中的作用奠定了基础。
Objective To construct recombinant shRNA lentiviral vector specific for shRNA p120 and to infect pancreatic cancer PANC-1 cells to identify the interference effects. Methods Three pairs of shRNA sequences were designed and synthesized against p120ctn mRNA and ligated into the linearized lentiviral vector pGCSIL-GFP. The PCR was identified and sequenced correctly and co-transfected with pGC-FU-p120ctn-3FLAG, a p120ctn gene-containing vector. 293T cells were screened for efficient shRNA sequences by Western blot analysis, packaged for lentivirus, and virus titers were determined. Recombinant lentivirus infected pancreatic cancer PANC-1 cells, real-time quantitative PCR and Western blot analysis to identify the interference effect. Results PCR and sequencing confirmed that the p120ctn-shRNA-LV lentiviral vector was successfully constructed and the titer reached 3×10 9 TU/ml. Real-time quantitative PCR revealed that the expression of p120ctn mRNA in PANC-1 cells was reduced by 82.6% (P<0.05) after recombinant lentivirus infection of PANC-1 cells. Western blot analysis indicated that the expression of p120ctn protein was significantly lower than that before transfection. Conclusion The p120ctn gene shRNA lentiviral vector was successfully constructed, which laid the foundation for the further study of the role of p120ctn in the invasion and metastasis of pancreatic cancer.