Construction of recombinant plasmid containing mouse vasoactive intestinal peptide gene and its expression in COS-7 cells / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To construct the eukaryotic expression plasmid containing mouse vasoactive intestinal peptide(VIP) gene with biological activities.</p><p><b>METHODS</b>VIP cDNA including the sequences of signal peptide was cloned from mouse thymus by RT-PCR, and then inserted into the mammalian expression vector pcDNA3.1 between Hind III and EcoR I restriction sites. COS-7 cells were transfected with pcDNA3. 1-VIP using liposome, the expression of VIP was identified by Western blot and ELISA. Supernatant of transfected cell culture was added to LPS-stimulated macrophages and the TNF-alpha production in cell medium was observed by ELISA.</p><p><b>RESULTS</b>The cloned VIP cDNA was confirmed by enzyme digestion and DNA sequencing. The expression of VIP was detected in the pcDNA3. 1-VIP transfected COS-7 cells by Western blot and ELISA. The VIP in culture supernatant potently inhibited TNF-alpha production by LPS-induced Macrophages in vitro.</p><p><b>CONCLUSION</b>The eukaryotic expression plasmid that expresses biological active murine VIP has been constructed successfully.</p>