Chronic Myeloid Leukemia Of Bcr / Abl Cdna Clone Of The Eukaryotic Expression Vector Construction,

Objective:To construct a eukaryotic expression vector pEGFP-bcr/abl and observe its expression in mammalian cell.Methods:Selecting the cell lines of K562. The gene encoding the P210 had been amplified by using reverse transcription polymerase chain reaction (PT-PCR) technique. The PCR products were purified. The purified products of bcr/abl were identified by digested with restricted enzymes EcoRâ… and BamHâ… . Both genes were sequenced to prove their authenticitieand. The recombinant plismid pEGFP-bcr/abl was transfected into mammalian cell lines of human COS-7 cells. After 72 hours'culture,we observe the COS-7 cells and analyzed the expressed protein by RT-PCR and Western-blot.Results:The size of RT-PCR product is 874, sequencing result revealed the sequence of amplified bcr/abl gene was identical with that in GeneBank. Restrictive enzyme (EcoRâ… and BamHâ… ) digestion analysis showed that the recombinant express plismid pEGFP-bcr/abl had been constructed successfully. The transfected COS-7 cells displaying green fluorescence were observed under fluorescence microscope. Western-blot analysis indicted that the molecular weight of the expressed protein of bcr/abl was about 28kDa, similar to the predicted value.Conclusion:The recombinant express plismid pEGFP-bcr/abl had been constructed successfully and could be expressed in mammalian COS-7 cells. The present study lay the foundation for the further tests of diagnose and therapy of CML by preparing monoclonal antibodies(McAb) against P210bcr/abl.