Cloning And Expression Of BCR-ABL Fusion Gene Fragment And Characterization Of Monoclonal Antibodies Against The Recombinant Protein

The BCR-ABL chimeric oncogene is caused by reciprocal translocation between long arms of chrosome 9 and 22, resulting in expression of a hybrid protein with constitutive active tyrosine kinase activity. Many studies had shown that p210^bcr-abl play the key role in the pathogenesis of chronic myeloid leukemia (CML). Therefore, detection of BCR-ABL fusion protein is mostly neccessary in the research and diagnosis of CML. Although p210^bcr-abl fusion region is regarded as the specific tumor marker, no monoclonal antibody binding the epitope of p210^bcr-abl fusion region is available, which already has become one of the bottlenecks of CML basic and clinical research. It arouses our interest that lacking monoclonal antibody against p210^bcr-abl fusion region is because of the absence of the B cell epitope for the fusion region or because of the particularity of preparation of its monoclonal antibody. The aim of this study is to develop the monoclonal antibody against p210^bcr-abl fusion protein and to provide a convenient and effective research tool for our laboratory.1. Predicting B cell epitopes of BCR-ABL fusion regionWe selected 151 amino acids flanking the fusion position according to the sequence of BCR-ABL chimeric protein. Parameters of hopp&Woods hydrophility,Janin accessibility,polarity,flexibility and secondary structure from BCR-ABL fusion region were analysed by softwares of EMBOSS and DNAstar.The results indicated that there were some B cell epitopes located in the fusion region of p210^bcr-abl. It suggested that antibodies targeted BCR-ABL fusion region would be produced in mice with immunization of specific synthetic peptide.2. Cloning, expression of BCR-ABL fusion gene fragment and purification and immunogenic analysis of the expressed proteinThe fragment of BCR-ABL fusion gene (b3a2) was amplified from a recombinant plasmid MIG210^bcr-abl by PCR and was cloned into pQE-31 vector according to the current open reading frame. This recombinant vector was transformed into E.coli M15 and be inducted by IPTG for expressing 6×His-tag protein p18^bcr-abl. The protein was purified and immunized the BALB/c mice for getting anti-serum. By indirect ELISA, the specific antibodies against the BCR-ABL fusion protein can be detected from the anti-serum. The results indicated that the recombinant protein p18^bcr-abl has immunogenicity against p210^bcr-abl and established the foundation for the further experimental study.3. Development and characterization of monoclonal antibodies against BCR-ABL fusion proteinAfter immunization with purified p18 ^bcr-abl fusion protein, hybridoma clones were produced by fusing spleen cells with myeloma cell SP2/0 at a ratio of 10:1 in polyethylene glycol 1450 according to standard procedure. Six strains of positive hybridoma clones were screened repeatedly by indirect enzyme-linked immunosorbent assay (ELISA) and subcloned by three times limiting dilution.Western-blotting analysis showed that Mabs could specifically bind the two protein bands from lysate of K562 cells which natively express p210^bcr-abl and c-abl protein. This suggested that the six kinds of monoclonal antibody, which were designated as abl-1A4, abl-2C5, abl-2H1, abl-4G2, abl-5F6, abl-5G4, respectively, specificially bind the abl region of BCR-ABL protein. Although we didn't obtain Mabs against BCR-ABL fusion region, the Mabs have the similar effect compared with purchased c-ab(l24-11)Mabs. The titers of these Mabs from ascitic fluids ranged from 1:103-1:106 detected by ELISA. After 8 months of storage in liquid nitrogen, all the cell lines retained their capabilities of secreting antibody. All Mabs have provided convenient, inexpensive tool for CML research in our laboratory.