Construction of chimeric gene expression vector inducing apoptosis of oral squamous carcinoma cells / 中国病理生理杂志

ABSTRACT

AIM: To gain cdcSTBX25A-fas chimeric gene bearing the regulative fragment cdcSTBX25A and opening read frame of fas in order to construct and identify eukaryotic expression vectors, pAdTrack-CMV-cdcSTBX25A-fas and pAdTrack-cdcSTBX25A-fas, which have the potential to transfer the tumor proliferative signal to promoting-apoptosis signal through up-regulate the fas expression by c-myc. METHODS: A pair of primers were designed according to the known sequences of cdcSTBX25A and fas. The cdcSTBX25A-fas chimeric gene was obtained by PCR reaction and inserted into the two plasmids pAdTrack-CMV and pAdTrack, respectively. The two recombinant plasmids were transferred into E. coli DH5?. The positive clones were screened in LB media with 50 mg/L kanamycin and identified by agarose gel electrophoresis, endonuclease digestion and PCR. The nucleotide sequence of inserted cdcSTBX25A-fas was determined by dideoxy chain termination method. Using software, BLAST was conducted to analyze the structure and sequence of the target fragments and compared with GenBank. RESULTS: The chimeric target gene, cdcSTBX25A-fas, of 1 603 bp was obtained. The positive host bacteria E. coli DH5? of recombinant plasmids were screened and amplified. The double-enzyme digestion showed the pAdTrack-CMV-cdcSTBX25A-fas and pAdTrack-cdcSTBX25A-fas presenting 9.2 kb, 1.6 kb bands, and 8.3 kb, 1.6 kb bands respectively. The sequence analysis confirmed that the two shuttle plasmids containing 1 597 bp cdcSTBX25A-fas with the ORF of fas. CONCLUSION: The eukaryotic expression plasmids pAd-Track-CMV-cdcSTBX25A-fas and pAdTrack-cdcSTBX25A-fas were successfully constructed.