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目的:构建Twist1的慢病毒真核表达载体,并建立稳定表达Twist1的乳腺癌细胞株,研究Twist1在乳腺癌中对上皮-间充质转变(EMT)的影响。方法:以乳腺文库为模板,PCR扩增Twist1编码序列,克隆到慢病毒p CDH载体,构建成p CDH-Twist1,转染293T细胞,Western印迹鉴定p CDH载体介导的Twist1的表达;包装病毒载体,感染ZR75-1细胞,用嘌呤霉素筛选得到稳定表达Twist1的细胞株,检测ZR75-1细胞对EMT的影响。结果:经Bam HⅠ、Eco RⅠ双酶切及测序证实得到p CDH-Twist1表达载体,Western印迹实验发现Twist1在293T细胞内表达;嘌呤霉素筛选得到稳定表达Twist1的ZR75-1细胞株。结论:构建了p CDH-Twist1的真核表达载体并建立了稳定表达Twist1的ZR75-1细胞,为进一步研究Twist1在EMT过程中的机制奠定了基础。
OBJECTIVE: To construct a lentiviral eukaryotic expression vector for Twist1 and to establish a stable breast cancer cell line expressing Twist1 to study the effect of Twist1 on epithelial-mesenchymal transition (EMT) in breast cancer. METHODS: The coding sequence of Twist1 was amplified by PCR from the breast cDNA library. The lentiviral vector pCDH was cloned into pCDH-Twist1 and transfected into 293T cells. The pCDH vector was used to detect the expression of Twist1. The vector was infected with ZR75-1 cells. The cells stably expressing Twist1 were screened with puromycin, and the effect of ZR75-1 cells on EMT was detected. Results: The pCDH-Twist1 expression vector was confirmed by double digestion with BamHI and EcoRI and confirmed by Western blotting. Twist1 was expressed in 293T cells. ZR75-1 cells stably expressing Twist1 were selected by puromycin. CONCLUSION: The eukaryotic expression vector pCDH-Twist1 was constructed and ZR75-1 cells stably expressing Twist1 were established, which laid the foundation for further study on the mechanism of Twist1 in EMT.