Construction of PCI-neo mammalian expression vector system containing murine 4-1BBL gene and its stable expression in hepatocellular carcinoma cell line / 中华肿瘤杂志

ABSTRACT

<p><b>OBJECTIVE</b>To study the construction of the PCI-neo mammalian expression vector system containing murine 4-1BBL gene and its stable and effective expression in rat hepatocellular carcinoma cell line CBRH7919.</p><p><b>METHODS</b>The murine full-length 4-1BBL cDNA was obtained and subcloned into the PCI-neo mammalian expression vector. The recombinant named as PCI-neo-4-1BBL was identificated by restriction enzyme digestion and sequencing. Subsequently, PCI-neo-4-1BBL was transfected into CBRH7919 with lipofectamine reagent, then G418-resistance clone was acquired and named as PCI-neo-4-1BBL-CBRH7919+. The stable expression of 4-1BBL mRNA in PCI-neo-4-1BBL-CBRH7919+ was detected by RT-PCR.</p><p><b>RESULTS</b>The fragment of 980 bp (4-1BBL) and 5.4 kb (PCI-neo) was shown after PCI-neo-4-1BBL had been digested by EcoR I and Not I and agarose gel electrophoresis. The DNA sequencing of 4-1BBL, proved to be identical to the data of 4-1BBL in Genebank, showed stable and effective expression in PCI-neo-4-1BBL-CBRH7919+.</p><p><b>CONCLUSION</b>The PCI-neo mammalian expression vector system containing murine 4-1BBL has been constructed successfully, which shows stable and effective expression in CBRH7919.</p>