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目的:构建血管生成素(ang)-2基因沉默载体,并研究其对肝癌hepG2细胞株的作用。方法:根据ang-2基因序列设计shRNA,并与载体pLVX-shRNA连接,转化感受态细胞,提取质粒测序鉴定。将慢病毒shRNA载体及其辅助包装原件载体质粒共转染293T细胞,在HEK293T细胞中标定其病毒滴度。用包装好的慢病毒以及慢病毒阴性对照感染靶细胞hepG2,感染成功后通过Western Blot方法评价干扰载体对靶基因的干扰效果,并采用MTT法检测其对靶细胞增殖率的影响。结果:酶切鉴定和测序结果证实成功构建了ang-2基因沉默载体。包装并浓缩慢病毒,滴度为1×10~8 TU/ml。将ang-2基因沉默载体感染hepG2细胞,倒置显微镜下可见绿色荧光。Western Blotting检测ang-2蛋白表达水平较对照组明显降低(P<0.05),而对hepG2细胞增殖率无明显影响。结论:成功构建了ang-2基因沉默载体并感染靶细胞hepG2,证实其对目的基因具有很好的沉默效果。
Objective: To construct ang-2 gene silencing vector and study its effect on hepG2 cell line. Methods: According to the sequence of ang-2 gene, shRNA was designed and ligated with vector pLVX-shRNA to transform competent cells. The plasmid was extracted and sequenced. 293T cells were co-transfected with the lentiviral shRNA vector and its auxiliary packaging vector and the virus titers were determined in HEK293T cells. The packaged recombinant lentivirus and lentivirus negative control were used to infect target cells hepG2. After successful infection, the effect of interfering vector on target gene was evaluated by Western Blot. The effect of target vector on MTT assay was tested by MTT assay. Results: Enzyme digestion and sequencing confirmed that ang-2 gene silencing vector was successfully constructed. The lentivirus was packaged and concentrated with a titer of 1 × 10 -8 TU / ml. HepG2 cells were infected with ang-2 gene silencing vector and green fluorescence was observed under inverted microscope. Western Blotting detected ang-2 protein expression was significantly lower than the control group (P <0.05), while hepG2 cell proliferation rate had no significant effect. Conclusion: ang-2 gene silencing vector was successfully constructed and infected target cell hepG2, which confirmed that it has a good silencing effect on the target gene.