| Interferon(IFN)is a host specific protein and has antiviral function in animal cell,which is secreted by some virus infection.Almost all of the animal cells produce type I interferon after suffers in viruses,bacteria,mycoplasma infection.The JAK-SATA cascade amplification system is activated by which IFNα of type I interferon couple with corresponding receptors in infected cells,and that also combine with IRTS,induces PKR,OAS and other protein expression,and inhibited virus replication.Therefore,Genetic engineering expression and purification of IFNα is of great significance for the antiviral infection of human and animal husbandry animals,and is expected to be a new way of treatment for livestock and poultry diseases.In this study,considering the significant difference of the codon between the prokaryotic and eukaryotic,thus according to the codon preference of Escherichia coli,the c DNA of procine interferon α(po IFNα)was optimized by codon optimization,so that it can more suitable for expression in E.coli.The codon optimized po IFNα c DNA was loaded onto the p UC57 cloning vector after entrustment company synthesis.The po IFNα c DNA was successfully subcloned into the intracellular expression vector p ET-22b(+)of E.coli by double enzyme digestion of Nde I and Xho I,and ligation in vitro.After genetically engineered bacteria were induced expression by IPTG and the fermentation bacteria were broken,meanwhile,the products were analyzed by SDS-PAGE,it showed that the recombinant porcine interferon(r Po IFNα)existed in the form of inclusion bodies,and identified with a molecular weight of 19.1k Da.By using the Image J software to analysis the strains with high expression,and selected them as fermentation strains for preservation.Through the optimization of fermentation conditions,the fermentation system was further amplified,and established 30 L fermentation tank culture.After induced expression 4 hours,the wet bacteria were obtained 80.15 g from every liter fermentation.From that,a large-scale and efficient production of recombinant protein r Po IFNα of fermentation process was initially established.After the fermentation engineering bacteria were broken,The r Po IFNα inclusion bodies underwent different concentration of Triton X-100 and urea washing experiment and dissolution experiment of lysated with different p H values,and thus set up a pretreatment process that r Po IFNα inclusion bodies were washed by using 4M urea washing solutions,and were dissolved by using 8M urea solution(p H=10.0)which containing 100 m M mercaptoethanol.The effects of two refolding methods and two redox systems on the refolding of Po IFNα were compared.Combined with the requirement of cost control,the process of Po IFNα was determined as a redox system with L-cystine and L-cysteine,and the rapid dilution method was used to regenerate Po IFNα,and the renaturation system was amplified to 100 L.A purification strategy has been designed which includes 50% saturation ammonium sulfate precipitation,CM-Sepharose ion-exchange chromatography,DEAE-Sepharose ion-exchange chromatography and Sephacryl S-100 molecular exclusion chromatography.And after that,the r Po IFNα with the purity more than 96% was obtained.The activity analysis of cytopathic inhibition assay showed that the activity of r Po IFNα was up to 8.04×106 U/ml after refolding and purification.After pilot production of three consecutive batches of r Po IFNα stoste,the results showed that the production and purification process of pilot production of r Po IFNα was relatively stable.In conclusion,this research has explored a process of fermentation and purification of po IFNα,which has stable production process,high purity and high specific activity,and this can provide reliable reference data and support for industrial production of po IFNα. |
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