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目的:利用慢病毒载体表达PES1基因,研究其对乳腺癌ZR75-30细胞生长的影响。方法:以乳腺文库为模板,PCR扩增PES1基因,克隆到pCDH载体,构建成pCDH-PES1,将其与包装载体共转293T细胞,包装成Lenti-PES1慢病毒载体并测定病毒滴度,感染乳腺癌ZR75-30细胞,Western印迹鉴定病毒载体介导的PES1蛋白的表达,并用生长曲线实验研究过量表达PES1对ZR75-30细胞生长的影响。结果:酶切和Western印迹表明得到pCDH-PES1阳性克隆,成功包装成Lenti-PES1慢病毒载体,滴度为2.5×107 pfu/mL;将此慢病毒载体感染ZR75-30细胞,Western印迹显示慢病毒载体成功表达PES1,生长曲线实验表明过量表达PES1可促进ZR75-30细胞的生长。结论:构建了PES1的慢病毒表达载体Lenti-PES1,在ZR75-30细胞中过量表达PES1可促进细胞的生长。
Objective: To express PES1 gene by lentiviral vector and study its effect on the growth of breast cancer ZR75-30 cells. Methods: The PES1 gene was amplified by PCR from the breast cDNA library and cloned into pCDH vector to construct pCDH-PES1. The recombinant plasmid was transformed into 293T cells with packaging vector and packaged into Lenti-PES1 lentiviral vector for virus titer and infection The expression of PES1 protein was detected by Western blotting in breast cancer ZR75-30 cells. The growth curve was used to study the effect of overexpression of PES1 on the growth of ZR75-30 cells. Results: The positive clones of pCDH-PES1 were confirmed by restriction enzyme digestion and Western blot. The positive clones were successfully packaged into Lenti-PES1 lentiviral vector with the titer of 2.5 × 107 pfu / mL. The lentiviral vector was used to infect ZR75-30 cells. The viral vector successfully expressed PES1, the growth curve experiments show that overexpression of PES1 can promote the growth of ZR75-30 cells. Conclusion: The lentiviral expression vector Lenti-PES1 of PES1 was constructed. Overexpression of PES1 in ZR75-30 cells can promote cell growth.