Objective To construct a eukaryotic expression vector of BPIFC gene with FLAG tag and establish stably transfected HaCaT cells.Methods The BPIFC gene fragment was amplified by PCR with primers and inserted into pcDNA3.1-flag-N eukaryotic expression vector by DNA recombination technology.The recombinant plasmid pcDNA3.1-BPIFC,after identified by restriction digestion and DNA sequencing,was transfected into HaCaT cells by PI Max linear transfection method.The stably transfected cell line was obtained by screening with G418,and the expression of BPIFC mRNA and protein were measured by RT-PCR and Western-blot,respectively.Results Restriction digestion and DNA sequencing revealed that the eukaryotic expression plasmid of pcDNA3.1-BPIFC was successfully construc-ted.RT-PCR results showed that the mRNA expression of recombinant plasmid was higher than that of blank load transfection group and blank control group.Western-blot results showed that BPIFC fusion protein was successfully ex-pressed in HaCaT cells.Conclusion The recombinant eukaryotic expression vector of pcDNA3.1-B PI FC has been constructed successfully and BPIFC protein fusion with FLAG affinity tag could be stably expressed for subsequent ex-perimental study.
维普
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