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目的克隆慢性髓系白血病(CML)融合基因bcr abl的cDNA序列 ,并在真核细胞中表达。方法以设计的两条引物扩增bcr abl融合位点两侧的cDNA ,测序后克隆至真核表达载体 pcDNA3.1 ,转染COS 7细胞。取常规培养的细胞进行RT PCR鉴定。结果①PCR扩增出490bp大小的cDNA ,其序列测定除第452位的碱基C突变为T外 ,其余序列均正确。②RT PCR法检测到转染细胞系中 ,有bcr ablmRNA的表达。结论成功地克隆了CML融合基因bcr abl融合位点两侧的490bp 序列并表达 ,为研制bcr abl基因疫苗治疗CML奠定了基础。
Objective To clone the cDNA sequence of bcr abl from chronic myeloid leukemia (CML) fusion gene and express it in eukaryotic cells. Methods Two cDNAs encoding bcr abl fusion sites were amplified by two designed primers. After sequencing, the cDNAs were cloned into eukaryotic expression vector pcDNA3.1 and transfected into COS7 cells. Routine cultured cells were identified by RT PCR. Results ① The 490bp cDNA was amplified by PCR. The sequence of the cDNA was confirmed to be correct except for the mutation of the base C at position 452 to T. ② The expression of bcr ablmRNA was detected in transfected cell lines by RT PCR. Conclusion The 490bp sequence on both sides of the bcr abl fusion site of the CML fusion gene was successfully cloned and expressed, which laid the foundation for the development of the CML gene therapy with the bcr abl gene vaccine.