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目的:构建重组慢病毒载体pWPT-ANXA2-Flag,并检测其在小鼠肝癌细胞系Hepa 1-6的表达情况。方法:利用DNA重组技术将人膜连蛋白A2(annexin A2,ANXA2)基因克隆入慢病毒表达载体pWPT中,通过酶切、测序验证后,将重组慢病毒载体pWPT-ANXA2-Flag与包装质粒pCMV-dR8.91、pCMV-VSV-G共转染人胚肾上皮细胞株293T,包装重组慢病毒LV-ANXA2-Flag,并感染小鼠肝癌细胞株Hepa 1-6,用RT-PCR检测ANXA2 mRNA转录水平,Western blot法检测ANXA2-Flag蛋白的表达情况,应用流式细胞仪检测细胞周期的变化。结果:经酶切及测序结果证实,构建了重组慢病毒载体pWPT-ANXA2-Flag;RT-PCR及Western blot结果显示慢病毒感染Hepa 1-6细胞后,ANXA2基因能够在细胞内正确的转录、翻译并稳定表达ANXA2蛋白;经过LV-ANXA2-Flag感染后,Hepa 1-6细胞S期细胞比例明显高于对照细胞。结论:成功建立ANXA2基因慢病毒表达载体pWPT-ANXA2-Flag,包装的慢病毒能够成功感染小鼠肝癌细胞系Hepa 1-6,并使ANXA2基因得以稳定表达,并通过ANXA2促进肿瘤细胞增殖。
OBJECTIVE: To construct recombinant lentiviral vector pWPT-ANXA2-Flag and detect its expression in mouse hepatoma cell line Hepa 1-6. Methods: The gene of annexin A2 (ANXA2) was cloned into lentiviral vector pWPT by DNA recombination technology. After digestion and sequencing, recombinant lentiviral vector pWPT-ANXA2-Flag was ligated with plasmid pCMV -dR8.91, pCMV-VSV-G were co-transfected into human embryonic kidney epithelial 293T cells and packaged with recombinant lentivirus LV-ANXA2-Flag and infected with mouse Hepa 1-6 hepatoma cell lines, and ANXA2 mRNA was detected by RT- The expression of ANXA2-Flag protein was detected by Western blot and the changes of cell cycle were detected by flow cytometry. Results: The recombinant lentiviral vector pWPT-ANXA2-Flag was constructed and confirmed by restriction analysis and sequencing. The results of RT-PCR and Western blot showed that ANXA2 gene could be transcribed correctly in Hepa 1-6 cells after lentivirus infection. Transforming and stably expressing ANXA2 protein; After infection with LV-ANXA2-Flag, the proportion of S phase cells in Hepa 1-6 cells was significantly higher than that in control cells. CONCLUSION: The recombinant lentiviral vector pWPT-ANXA2-Flag of ANXA2 gene is successfully constructed and the packaged lentivirus can successfully infect mouse hepatoma cell line Hepa 1-6. The ANXA2 gene can be stably expressed and the proliferation of tumor cells can be promoted by ANXA2.