Expression And Antiviral Activity Analysis Of The Goose Interferon-alpha Amino Acids With No Redundancy In Pichia Pastoris

Interferon (interferon,IFN) is a glucoprotein which secreted by the cells with a broad-spectrumanti-viral, anti-cell division, immunomodulatory activity and in different ways affect cell metabolism,growth. In the type of interferon alpha interferon broad-spectrum antiviral ability is the most significant.Pichia expression system is widely used one of exogenous protein in eukaryotic expression system. Atpresent, the use of the system has been successfully expressed the animals such as mammals, birds, fishinterferon. Therefore, the pichia expression system mass production has the biological activities ofinterferon has a broad appprospect.In order to get Goose interferon-alpha (GoIFN-α) with no redundancy amino acids and antiviral activities. GoIFN-α was amplified by PCR from Goose genome that extracted from fresh liver, Productsof PCR consistent with GoIFN-α(GenBank:AY524422) nucleotide sequence. The gene and the α-factorsignal sequence of pPICZαA were amplified by Fusion PCR. Atfer the first code near α factor weremodified to the hobby of Piehia Pasotrsi ye-ast the gene of GoIFN-α was inserted into PPICZαA.Theresulted recombinant expression vector was linearized with Sac Ⅰand then transformed into PichiaPastorsi GS115and X33yeast strain to integrate into yeast genome.After recombinant yeast strainswere indueed by methanol for72hours, the expression conditions of the best tirne and methanolconeentration were optimized. SDS-PAGE showed that expression product was about25~35ku. Therecombinant protein is larger than the theoretical value(18ku). The expressed protein was confirmed asGoIFN-α by Western-blot. The No.7recombinant yeast were picked as highest expression of strain byDot-ELISA.To improve the reorganization GoIFN-alpha protein expression of the quantity, No.7yeast waschoose to conduct fermentation tank culture. The values of dissolved oxygen and the amount ofmethanol were controled in the process of fermentation. SDS-PAGE showed that expression product was about25~35ku.The antiviral aetivity was evaluated by using GPMV/VSV on duck embryo fibroblast.The resultshowed that the GoIFN-α have cytokine activity.The recombinant GoIFNα could inhibit theGPMV/VSV on duck embryo fibroblast and had an antiviral activity of1.80×10~5U/mg/2.12×10~5U/mg.Expression the highest expression of recombinant yeast in fermentation tank. The recombinant GoIFNαcould inhibit the GPMV/VSV on duck embryo fibroblast and had an antiviral activity of2.7×10~5U/mg/4.21×10~5U/mg.