Source
Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fourth Military Medical University, Xi'an 710032, China. lixia7305@yahoo.com.cn
Abstract
AIM:
To construct the chimeric cblN/Zap molecule, and to observe its effect on TCRzeta protein in pcDNA3.1(+)-cblN/Zab transfected Jurkat cells.
METHODS:
Total RNA of Jurkat cells were isolated and reversely transcribed into cDNA, which were used as templates to amplify Zap SH2 by PCR. cblN gene tagged with 24 bp flag was amplified by PCR using pEFHAcbl plasmid encoding human cbl as templates. BamH I and EcoR V restriction enzyme digestion sites were introduced into flank SH2 in pcDNA3.1(+) cblN by overlapping extension PCR. Then pcDNA3.1(+)-cblN/Zap was obtained by replacing SH2 of cblN with ZapSH2. After confirmation by enzyme digestion and sequencing, the recombined vector was stably transfected into Jurkat cells with lipofectin. The expression of flag-cblN/Zap was detected by RT-PCR and Western blot, and its effect on TCRzeta protein was analyzed by Western blot.
RESULTS:
Restriction enzyme digestion analysis of the pcDNA3.1(+)-cblN/Zap recombinant vector showed that the expected fragments were produced and confirmed by sequencing. pcDNA3.1(+)-cblN/Zap was stably transfected into Jurkat cells and the expression of flag-cblN/Zap in the stable clones was confirmed by both RT-PCR and Western blot. The expressed cblN/Zap down-regulated TCRzeta protein in Jurkat cells.
CONCLUSION:
Chimeric cblN/Zap is able to down-regulate TCRzeta in Jurkat cells.