慢性髓系白血病bcr—abl融合基因在真核细胞中的表达

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468bp的cDNA，测序正确后，将其克隆至真核表达载体pcDNA3.1通过电穿法转染至p815细胞中，应用G418筛选阳性细胞克隆，在含10％FCS的RPMI1640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468bp的cDNA，序列测定除第448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 EXPRESSION OF CHRONIC MYELOGENOUS LEUKEMIA bcr-abl FUSION GENE IN EUKARYOTE

Chronic myelogenous leukemia bcr abl fusion gene was cloned and a recombinant expression vector was constructed and expressed in p815 cells to explore the feasibility of bcr abl fusion gene in CML gene immunization therapy. Two primers were designed for the amplification of a 468bp bcr abl cDNA coding sequence by PCR. After the PCR product was confirmed by the sequence analysis, it was properly inserted into the expression vextor pcDNA 3.1. The recombinant vector was transfected in p815 cells by means of electric perforation. These stable expression cell colonies were obtained by G418 selection ,and its expression was confirmed by RT PCR after cultivation in RPMI 1640. A 468bp bcr abl cDNA was amplified by PCR, and sequence analysis showed the whole correct sequence except base C mutated for T in position 452. A recombinant expression vector was constructed, and it could be tranfected into p815 cells by electric perforation. RT PCR examination showed that bcr abl had been transcribed in cell clones.