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


CONCLUSIONS
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.
Li
Chuang
C
Institute of Hematology, Medical College of Jinan University, Guangzhou 510632, China. stlichuang@163.com
Wu
Xiu-li
XL
Li
Yang-qiu
YQ
Zhou
Yu-bing
YB
Chen
Shao-hua
SH
Yang
Li-jian
LJ
Zhu
Kang-er
KE
D056890:Eukaryota / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D011948:Receptors, Antigen, T-Cell / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D011948:Receptors, Antigen, T-Cell