bcr-abl基因真核表达载体的构建及其在COS-7细胞中的表达

目的 构建慢性粒细胞白血病bcr-abl融合基因的真核表达载体,并在哺乳动物细胞COS-7中高效表达.方法 提取慢性粒细胞白血病K562细胞的RNA,经RT-PCR技术扩增得到bcr-abl基因后,将其克隆入真核表达载体pEGFP-N3中,并进行PCR和酶切鉴定.鉴定为阳性的克隆,再进一步进行测序分析.将构建成功的真核表达载体转入COS-7细胞中进行瞬时表达.结果 反转录PCR扩增后的产物,经琼脂糖凝胶电泳可以在约874 bp处看见一特异性的条带,序列分析证明扩增产物与GeneBank中的bcr-abl基因序列相同.将pEGFP-bcr-abl真核表达载体转入COS-7细胞后,经RT-PCR、Western blotting检测证明表达产物正确.结论 成功构建了含有bcr-abl融合基因的真核表达质粒,从而为进一步研究其结构和功能,寻找CML诊疗的新方法奠定了基础.

Construction of bcr-abl gene eukaryotic expressing vector and its expression in COS-7 cells

Objective To clone and construct eukaryotic expressing vector of bcr-abl fusion gene and to express the gene in the mammal COS-7 cell lines. Methods bcr-abl fusion gene was amplified from human chronic myeloid leukemia (CML) K562 cell lines by RT-PCR and the fragment of cDNA was retrieved,purified and cloned into the pEGFP-N3 eukaryotic expressing vector. After the selection of the positive clone and by restriction enzyme analysis and DNA sequencing, the correct plasmid was transfected into COS-7 cell lines and observed the transient expression. Results A 874 bp DNA fragment was amplified by RT-PCR. The sequence analysis showed it was consistent with bcr-abl gene of GeneBank. RT-PCR, Western blotting analysis provided strong evidences that bcr-abl gene was expressed successfully in transfected COS-7 cells.Conclusion The eukaryotic expressing vector of bcr-abl fusion gene was constructed, it will lay the foundation for further study of bcr-abl gene in the diagnosis and treatment of CML.