Expression And Antiviral Activity Analysis Of The Recombinant Canine IFN-α7in Ecoil And Pichia Pastoris Yeast

Interferon (interferon, IFN) is a glucoprotein which secreted by the cells with a broad-spectrum anti-viral, anti-cell division, immunomodulatory activity and in different ways affect cell metabolism, growth. Recently, people are more and more concerned about the effects of interferons on disease prevention and cure of poultry, because of the characters of non-medicine and non-toxic side effects. However, the research and application of CaIFN-α is behindhand relatively.1To achieve high-level expression of soluble recombinant Canine Interferon-alpha-7in E.coli, we had merged the Ssp DnaB intein (SDI) coding sequence into the upstream of the Canine Interferon-alpHa cDNA, and then cloned it into the expression vector pMAL-c2X. Finally, we construct the Intein expression plasmid, named as pMAL-c2X-SDI-CaIFN-a The recombinant expression plasmids were transformed into E.coli Rosetta and induced by IPTG. The result showed that the fusion protein was expresse solubly and more pure proteins were produced by Amylose Resin column purification. The purified protein shaved the antiviral activity in MDCK/CDV system. The rCaIFN-α activity was3.5×105U/mg.2The gene of canine interferon alpha was modified to get the recombinant portein with natural structure. Atfer the first code near a factor were modified to the hobby of Piehia Pasotrsi yeast the gene of CaIFN-α was inserted into Pichia Pastorsi yeast expression vector PPICZaA.The resulted recombinnatexpression vector was linearized with Sac I and then transformed into Pichia Pastorsi GS115yeast strain by LiCl to integrate into yeast genome and obtained engineering Piehia Pasrorsi srtain GS115-pPICZaA-CaIFN-a. SDS-PAGE showed that expression product of the gene of Cnaine’s mature interferon-alpha in culture supernatant of GS115-PPICZaA-CaIFN-a was about25kDa. Because there were two glycosation sites which led to theglycosation of expressed product.In addition, the target protein was added to MDCK, which were subsequently infected by the100TCID50CDV. The result showed the good cytokine activity.The recombinant CaIFNα activity was9.6×106U/mg. Expression the highest expression of recombinant yeast in fermentation tank. The recombinant CaIFNα activity was2.1×106U/mg.