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目的:构建4E-BP1及其T37A、T46A、S65A、T70A突变体4E-BP1-4A基因表达的重组慢病毒载体,研究其对胃癌HGC27细胞生长的影响。方法:PCR扩增4E-BP1基因及其突变体4E-BP1-4A基因并克隆到pCDH载体,构建成pCDH-4E-BP1、pCDH-4E-BP1-4A,将其与包装载体共转染293T细胞,包装成Lenti-4E-BP1及Lenti-4E-BP1-4A重组慢病毒载体,将此慢病毒感染胃癌HGC27细胞,Western印迹鉴定病毒载体介导的4E-BP1、4E-BP1-4A蛋白的表达,MTT、克隆形成和软琼脂方法研究过量表达4E-BP1、4E-BP1-4A对胃癌HGC27细胞生长的影响。结果:包装成Lenti-4E-BP1及Lenti-4E-BP1-4A重组慢病毒载体,并将此慢病毒载体感染胃癌HGC27细胞;MTT、克隆形成、软琼脂实验表明过量表达4E-BP1可抑制胃癌HGC27细胞的生长,过量表达4E-BP1-4A时抑制效果更明显。结论:构建了4E-BP1、4E-BP1-4A的重组慢病毒表达载体,在胃癌HGC27细胞中过量表达4E-BP1可抑制细胞生长,过量表达4E-BP1-4A的抑制效果更明显。
OBJECTIVE: To construct recombinant lentiviral vector expressing 4E-BP1 and its 4E-BP1-4A gene of T37A, T46A, S65A and T70A and study its effect on the growth of gastric cancer HGC27 cells. Methods: The 4E-BP1 gene and its mutant 4E-BP1-4A gene were amplified by PCR and cloned into pCDH vector to construct pCDH-4E-BP1 and pCDH-4E-BP1-4A. The recombinant plasmid was co-transfected with 293T Cells were packaged into Lenti-4E-BP1 and Lenti-4E-BP1-4A recombinant lentiviral vector. The lentiviral vector was used to infect gastric cancer HGC27 cells. Western blotting was used to identify the viral vector-mediated 4E-BP1 and 4E-BP1-4A proteins The effects of overexpression of 4E-BP1 and 4E-BP1-4A on the growth of gastric cancer HGC27 cells were studied by MTT assay, colony formation assay and soft agar assay. Results: Lenti-4E-BP1 and Lenti-4E-BP1-4A recombinant lentiviral vector were packaged and infected with this lentiviral vector in HGC27 cells. MTT, colony formation and soft agar assay showed that overexpression of 4E-BP1 inhibited gastric cancer HGC27 cell growth, over-expression of 4E-BP1-4A inhibitory effect is more obvious. CONCLUSION: Recombinant lentiviral vector expressing 4E-BP1 and 4E-BP1-4A has been constructed. The overexpression of 4E-BP1 in gastric cancer HGC27 cells can inhibit cell growth. The inhibitory effect of 4E-BP1-4A overexpression is more obvious.