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[Construction of recombinant plasmid containing mouse vasoactive intestinal peptide gene and its expression in COS-7
Song-zhao Zhang1, Jian-gen Shen, Zhi-xiong Liao
1Institutes of Immunoology, College of Medicine, Zhejiang University, Hangzhou 310031, China.
Objective:
To construct the eukaryotic expression plasmid containing mouse vasoactive intestinal peptide(VIP) gene with biological activities.
Methods:
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.
Results:
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.
Conclusion:
The eukaryotic expression plasmid that expresses biological active murine VIP has been constructed successfully.
