The Construct Of CML Gene Vaccine And Establishment SP2/O Cell Lines Expressing Bcr-abl Fusion Gene Fragment

The crucial genetic event in Chronic myeloid leukemia is the generation of a t (9;22)(q34;qll) reciprocal chromosomal translocation in a hematopoietic stem cell. This translocation creates a new gene, bcr-abl on the 22q" or Ph chromosome, and the reciprocal abl-bcr on the derivative 9q+. The final product of this genetic rearrangement is a 210 kDa cytoplasmic fusion protein, or p210 bcr"abl. The bcr-abl protein is leukemogenic because its Abl-derived tyrosine kinase is constitutively activated. This leads to malignant transformation by interference with basic cellular processes. The chimeric p210 fusion protein is a potential antigen as the junction regions contain a sequence of amino acids that is not expressed on any normal cell. Therefore, the unique sequence can be considered truly as a tumor specific antigen of CML. The study focused on the bcr-abl fusion gene vaccine, and consisted of three sections as the following:1. Cloning and expression of CML bcr-abl fusion gene fragment in proeukaryotesThe 450 bp of bcr-abl fusion gene was amplified from total RNA of K562 cells by RT-PCR and was cloned into the dowmstream of GST gene in pGEX-6P-l vector. The recombinant vector was transformed into E.coli BL2i cells for expression. Fusion protein was 42 KD identified by SDS-PAGE. The ICR mice were immunized with the fusion protein. The polyclone antibody was combined to K562 cells which express p210 bcr-abl through indirect immune fluorescence immunoassay. This result suggested that the fusion protein had specific immunogenicity of p210 bcr-abl.2. Molecular cloning and expression of CML bcr-abl fusion gene fragment in eukaryotesBased on the gene sequence encoding bcr-abl (b3a2) fusion gene from chronic myelgenous leukemia K562 cell line, we designed a pair of primers, obtained the 450bp gene fragment of bcr-abl by using RT-PCR, and inserted the gene into plasmid pDNA3.1 via pGEM-T easy vector to construct recombinant plasmid. The recombinant plasmid pcDNAbcr-abl was transfected into CHO cells with liposome. The recombinant plasmid was transiently expressed the fragment of bcr-abl fusion protein hi cytoplasm of CHO cells confirmed by indirect immune fluorescence immunoassay. The BALB/c mice inoculated with pcDNAbcr-abl by intramusular injection could elicit to produce corresponding antibodies to K562 cells expressing p210 bcr-abl.3. Establishment chimeric human-murine tumor cell lines which satably expressed bcr-abl fusion fragmentThe bcr-abl fusion gene fragment was subcloned into retroviral vector pLXSNfrom pGEMbcr-abl. The recombinant retroviral vector pLXSNbcr-abl was transfected into PT67 packaging cells with the help of lipofectamine. The positive clones were selected out and cultured after G418 selection. Then viral supernatant was collected. The SP2/0 cells were infected with the collected viral supernatant. After G418 selection, we observed that the bcr-abl fusion gene fragment was integrated into the chromosome of SP2/0 cells infected with recombinant retrovirus stably confirmed by PCR and expressed in SP2/0 cells confirmed by RT-PCR respectively. Chimeric human-murine tumor cell lines expressing the fragment of bcr-abl fusion protein were established and would be used as experimental cell model for anti-CML immunotherapy.