[Construction of recombinant plasmid containing mouse vasoactive intestinal peptide gene and its expression in COS-7 cells].
To construct the eukaryotic expression plasmid containing mouse vasoactive intestinal peptide(VIP) gene with biological activities.



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.



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.



The eukaryotic expression plasmid that expresses biological active murine VIP has been constructed successfully.
Zhang
Song-zhao
SZ
Institutes of Immunoology, College of Medicine, Zhejiang University, Hangzhou 310031, China.
Shen
Jian-gen
JG
Liao
Zhi-xiong
ZX
Pan
Jian-ping
JP
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014660:Vasoactive Intestinal Peptide / Q000096:biosynthesis / Q000235:genetics*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014660:Vasoactive Intestinal Peptide