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[Construction and expression of idiotype TCR Valpha1-pIRES-TCR Vbeta8 eukaryotic vectors in vitro].

AIM
To construct the eukaryotic vectors with idiotype TCR Valpha1-pIRES-TCR Vbeta8 of Jurkat cell line and investigate their expression in vitro after transferred into eukaryotic cells.


METHODS
TCR Valpha and Vbeta subfamilies of Jurkat cells were analyzed by using RT-PCR and genescan technique. Then the monoclonal TCR Valpha1 and Vbeta8 genes of Jurkat cells were cloned into multiple clone site (MCS) A and MCS B of the eukaryotic vector pIRES respectively. Its sequences were identified by restriction enzyme cutting and sequence analysis. The expression of TCR mRNA and idiotypic protein in transferred A549 and Molt4 cells was tested by RT-PCR, indirect immunophenotyping fluorescein dyeing and flow cytometry, respectively.


RESULTS
Two recombinavot eukaryotic vectors with idiotype TCR Valpha1-pIRES-TCR Vbeta8 were developed successfully and the expression of both idiotypic TCR mRNA and protein was detected in transferred A549 and Molt4 cells.


CONCLUSION
The successful construction of the two eukaryotic vectors with idiotype TCR Valpha1-pIRES-TCR Vbeta8 will provide a basic method for further study of the specific TCR gene modified T cells.