| Vasoactive intestinal peptide (VIP) is a 28 aminoacid peptide originally isolated and described by Said and Mutt in lung and small intestine in its capacity as vasodilator, with a highly conserved sequence in vertebrates. Recently, this pleiotropic neuropeptide has been rediscovered in the immune system, and it has been identified as a potent anti-inflammatory factor and immunomodulators. As for downregulating both inflammatory and autoimmune Components, VIP might extend the range of therapeutic treatments available for various disorders, including acute and chronic inflammatory diseases, septic shock and autoimmune diseases.Some evidence indicate that treatment of arthritic mice with VIP decreases the frequency and severity of arthritis, which emerged as a very attractive candidate for the treatment of arthritis. VIP and VEP analogues may be demonstrated as a promising alternative treatment for septic shock, but also states that there could be some pitfalls in this approach, such as side effects to chronic administration of VIP, including detrimentalgastrointestinal effects and a general immunosuppression, largely limited by the rapid enzymatic degradation in vivo. VIP gene transfer could offset these disadvantage, but has been not reported yet.More and more study indicate that cell- or tissue-specific gene delivery to treat inflammatory diseases and autoimmune diseases will be a key direction of future research. Foreign researcher address that IL-la gene to treat RA in both animal model and phase I clinical trials was efficiently completed. So the objective of the project is constructing eukaryotic expression of plasmid pcDNA3.1-vip, and studying the biological activity of VIP expressed in vitro, to prepared with advanced animal experiment and clinical trial.Methods1 .Amplification of target gene: The cDNA encoding VIP was obtained by RT-PCR from total RNA of murine thymusocytes using Trizol method extraction. Primers designed with Hind III and EcoR I endonuclease sites were used in PCR to amplify VIP-28 gene(include signal petide gene sequence), Amplified fragment was purified.2. Construction of eukaryotic expression vector: Having been digested by restriction enzymes Hind III and EcoR I, VIP-28 fragment and linearized expression vector pcDNA3.1 were ligated to construct recombinant eukaryotic expression vector named as pcDNA3.1-VIP which then was identified by Hind III and EcoR I digestion as well as sequencing.3. Transient expression of pcDNA3.1-VIP in cos-7 cells: cos-7 cellswere transfected with pcDNA3.1-VIP by Liposome.after the 48h, collected the liquid cultures, lysed the cells using a sonicator, and centrifuged to separate supernatant and pellet. At last, analysized the products by ELISA and Western blots.4. Study of biological activity of expressed protein: diverse volume supernatants were injected into the culture medium of Macrophge with LPS-stimulated, observed the dose-response for the inhibitory effect of TNF-a production, including the effect as joined the neutralize antibody.ResultsThe cDNA encoding VIP-28 which was obtained by RT-PCR from VIP mRNA of murine thymusocytes had about 160bp expected. When digested by restriction enzymes separately, VIP-28 fragment and linearized expression vector pcDNA3.1 were ligated to have succeeded in constructing recombinant eukaryotic expression vector pcDNA3.1-VIP. It was identified by restriction enzyme digestion and sequencing, showed the fragment VIP-28 inserted with signal peptide sequence and the identical 28 ammo acid by translation.Through transfect into COS-7 cells by liposome, separate supernatant and pellet using a sonicator, ELISA and Western blots showed that the VIP-28 secreted from the transfected cells was also found in the supernatants Which had excellent biological activity for the dose-inhibitory effect of TNF-a production of Macrophge with LPS-stimulated and for the disappeared inhibitory effect when Neutralized by antibody.Conclusions1 .Recombinant eukaryotic expression vector...
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