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克隆慢性髓系白血病 (CML)融合基因bcr abl融合位点周围的cDNA序列 ,构建重组真核表达载体并表达于 p815细胞 ,探索bcr abl基因免疫治疗CML的可行性。设计两条引物扩增bcr abl融合位点周围 46 8bp的cDNA ,测序正确后 ,将其克隆至真核表达载体pcDNA 3.1,通过电穿孔法转染至 p815细胞中 ,应用G418筛选阳性细胞克隆 ,在含 10 %FCS的RPMI16 40中常规培养 ,收集细胞进行RT PCR鉴定。结果PCR扩增出了可用于基因免疫的位于bcr abl融合基因位点周围的 46 8bp的cDNA ,序列测定除第 44 8位碱基C突变为T外其余序列均正确 ;构建了重组真核表达载体 ,并用电穿孔法将其转染 p815细胞 ,RT PCR法检测到该细胞系有bcr ablmRNA水平的表达
The cDNA sequence surrounding the bcr abl fusion site of the chronic myeloid leukemia (CML) fusion gene was cloned to construct a recombinant eukaryotic expression vector and expressed in p815 cells. The feasibility of immunotherapy for CML by bcr abl gene was explored. Two primers were designed to amplify the 46 8 bp cDNA surrounding the bcr abl fusion site. After sequencing was correct, it was cloned into the eukaryotic expression vector pcDNA 3.1 and transfected into p815 cells by electroporation. The positive cell clone was screened by G418. Cells were routinely grown in RPMI1640 with 10% FCS and cells were collected for RT PCR identification. RESULTS: The 46 8 bp cDNA which was located around the bcr abl fusion gene locus was amplified by PCR and used for gene immunization. The sequences were correct except that the 44th base C mutation was T, and the recombinant eukaryotic expression was constructed. The vector was transfected into p815 cells by electroporation. The expression of bcr abl mRNA in the cell line was detected by RT PCR.