Construction Of The Clone Containing Recombinant Vasoactive Intestinal Peptide And Evaluation On Its Biological Activity

Vasoactive intestinal peptide(VIP)has various biological functions,including the stimulation of the gastrointestinal tract,neuropeptide function and immune function.It plays its main physiological roles on the gastrointestinal tract,cardiovascular system,immune system,and inhibits tumor growth.However,VIP degrades rapidly in the body,its half-life in vivo is less than one minute,which extremely restricts its clinical application.The current methods used to improve the half-life in vivo of VIP are all unsatisfactory.It is urgent to find an economical,efficient and secure way to extend the half-life in vivo of VIP.HSA as a carrier protein,can be effective in prolonging the half-life of small proteins in the blood circulation.We use the pcDNA3.1-HSA plasmid as a template,HSA gene is obtained by PCR.The fully synthetic VIP gene is fuse to HSA gene by a linker,then inserts into pcDNA3.1 to get VIP recombinant protein expression vector of pcDNA3.1-HSA-VIP.Convert it into the top 10 competent cell to sequencing.After sequenced,the clone with entirely correct DNA sequence in the fungus is then expand after incubation for extraction of plasmid DNA.Then transfect it into CHO cells for expressing the desired recombinant protein in the cell supernatant.VIP has anti-inflammatory activity,and can significantly abrogate the increase of pro-inflammatory cytokines of serum resistin by LPS-induced acute inflammation state.Accordingly,we test the resistin levels to represent the biological activity of recombinant VIP protein by serum resistin ELISA kit.Experimental results show that the concentration of resistin in sample VIP+LPS group is 0.04 ?g/L,which is significantly lower than that in LPS 50 ?g/g group(1.40 ?g/L)and LPS 5 ?g/g group(1.53 ?g/L),suggesting that VIP suppresses the increase in resistin induced by LPS.However,resistin concentration is 3.17 ?g/L in the saline group,it is far below the normal values reported in the literature.Evidences show that it is due to the poor sensitivity of the kit.Further research is still in progress.Behavioral analysis in mice indicates that VIP recombinant protein had some certain biological activity.