Analysis Of Bcr-abl Associated T-cell Receptor Vα/Vβ Subfamily Gene Repertoire And Construction Of Idiotype TCR Vα/Vβ Eukaryotic Vector

Objective: The first aim of this study was to investigate bcr-abl associated T cell receptor (TCR)Vαand Vβgene repertoire by analyzing the restricted usage and clonal expansion of TCRVαand Vβsubfamily T cells from the normal individual peripheral blood induced bybcr-abl peptideso The second aim was to construct the idiotype TCR Vα-PIRES-TCR Vβeukaryotic vector and investigate its expression in vitro. The data will be expected toprovided the methods in development of the leukemia antigen specific cytotoxic Tlymphocyte (CTL) modified by the specific TCR.Motheds: T-cells (HLA-A0201+) were induced and amplified by IL-2, anti-CD3 and anti-CD28antibody with synthetic bcr-bal peptide (KQSSKALQR, 50μg/mL) in vitro by liquid T-cellculture. The induced T cells were collected at different time points after culture (10th and18th day). The expression and clonality of TCR Vαand Vβsubfamilies within induced Tcells were analyzed by using RT-PCR and genescan technique. The eukaryotic vector withidiotype TCR Vα1-pIRES-TCR Vβ8 of Jurkat cell TCR Vα1 and Vβ8 subfamily wasconstructed by the technology of gene cloning as well. Its sequences were identified byrestriction enzyme cutting and sequence analysis. Both the TCR mRNA and idiotypicprotein expression were tested by RT-PCR, indirect immunophenotyping fluoresceindyeing,Flow Cytometry,dot-blotting,SDS-PAGE-western-blot in transferred A549,K562 and Molt4 cells respectively.Results: (1) The clonality of TCR Vα11,Vβ22,Vβ24 subfamilies T cells were changed frompolyclone to oligoclonal tendency, indicating that the restricted and cloanl expansion ofthese three TCR Vα11,Vβ22 and Vβ24 subfamilies might be related to the induction ofbcr-abl peptide (KQSSKALQR).(2) Two idiotype TCR Vα1-pIRES-TCR Vβ8 recombinant eukaryotic vectors weredeveloped successfully, and the expression of both idiotypic TCR mRNA and proteincould be detected in transferred A549,K562 and Molt4 cells.Conclusion: The character of the TCR Vαand Vβrepertoire and cloanl expansion of the normalindividual T-cells (HLA-A0201+) could be identified after induced by the bcr-abl peptide.The idiotype TCR Vα1-pIRES-TCR Vβ8 eukaryotic vectors were developed successfully, it would be provided the basic data for the study on specific TCR gene modified T cells.