Construction of recombinant plasmid containing mouse vasoactive intestinal peptide gene and its expression in COS-7 cells.
- Author:
Song-zhao ZHANG
1
;
Jian-gen SHEN
;
Zhi-xiong LIAO
;
Jian-ping PAN
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Base Sequence; COS Cells; Cercopithecus aethiops; Cloning, Molecular; DNA, Complementary; genetics; Eukaryotic Cells; metabolism; Mice; Molecular Sequence Data; Plasmids; Recombinant Proteins; biosynthesis; genetics; Recombination, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Vasoactive Intestinal Peptide; biosynthesis; genetics
- From: Journal of Zhejiang University. Medical sciences 2005;34(2):148-151
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct the eukaryotic expression plasmid containing mouse vasoactive intestinal peptide(VIP) gene with biological activities.
METHODSVIP 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.
RESULTSThe 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.
CONCLUSIONThe eukaryotic expression plasmid that expresses biological active murine VIP has been constructed successfully.
