摘要
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 and identify combinant lentiviral vectors for overexpression of 4E-BP1 and the T37A, T46A, S65A, T70A mutant of 4E-BP1-4A, and to study their effects on the grown of the gastric can- cer cell HGC27. Methods： 4E-BP1 and 4E-BP1-4A genes were amplified using PCR and cloned into the pCDH vector. The resulting plasmid pCDH-4E-BP1 and pCDH-4E-BP1-4A with the packing carrier mix were co-trans- letted into 293T cells to obtain packaged lentivirusparticles, and then the packaged lentiviruspartieles were used to infect HGC27 cells. Western blot was performed to identify lentivirus-mediated expression of 4E-BP1 and 4E-BPI-4A in HGC27 cells. MTT and colony-formation assays were performed to study the effect of overexpres- sion of 4E-BP1 and 4E-BP1-4A on the grown of HGC27 cells. Results： The recombinant lentivirus expressed 4E-BP1 and 4E-BP1-4A efficiently in HGC27 cells. The MTT and colony-formation assays indicated that overex- pression of 4E-BP1 and 4E-BP1-4A inhibited the growth of HGC27 cells. Conclusion： Recombinant lentiviruses overexpressing 4E-BP1 and 4E-BP1-4A were successfully constructed. Overexpression of 4E-BP1 and 4E-BP1- 4A suppressed the growth of HGC27 cells.
出处
《生物技术通讯》
CAS
2013年第2期193-196,共4页
Letters in Biotechnology
基金
国家自然科学基金(30770834
30870961)
国家高技术研究发展计划(2008AA02Z123)
