Establishment Of Porkaryotic Expression System And Bioactivity Analysis Of Porcine IFNα1-THYα1Fusion Proteins

Interferon(IFN), a cytokine, possesses antiviral, anticancer and immunomodulatoryeffects. Porcine interferon alpha1（poIFNα1） possesses a broad spectrum of antiviraleffects with fewer side-effects and higher efficience. Thymosin alpha1（THYα1）, a28-amino acid peptide, plays a crucial role in the differentiation and maturation ofT-cell and enhancement of NK-cell and immune responses. Antiviral effect ofcombination of IFN and THY rises dramatically in comparison to IFN or THYmonotherapy. Currently, the clinical veterinary lacks highly efficient and low-pricedantiviral biologics. In spite of their immune effects, IFN and THY have little clinicalveterinary application. The fusion expression of poIFNα1and THYα1has not beenreported. In order to enhance antiviral effects and lower production cost, the fusiongenes of poIFNα1and THYα1were constructed and expressed in this study, whichprovided a new method to prevent and treat porcine viral diseases.Codon bias plalys a important role in the expression level of extrinsic protein inEscherichia coli, so the poIFN-α1and THYα1genes were optimized and synthesizedon the basis of the genic sequence of poIFN-α1in Genbank，the protein sequence ofTHYα1and the codon bias in Escherichia coli. In order to express thepoIFNα1-THYα1fusion proteins and maintain their bioactivities, three differentlinkers, ARG（LinkerA），GGGGSARGGGGGS（LinkerB），GGGGSGGGGSGGGGS（LinkerC），were designed between poIFNα1and THYα1by SOE-PCR. The ARGpeptide sequence is one of recognition sequences for thrombin protease and theGGGGS peptide has a high flexibility to help to maintain the bioactivities of fusionproteins. The poIFNα1gene and three different poIFNα1-THYα1fusion genes werecloned into pRSFDuet-1expression plasmids respectively, the identification resultsindicated that the expression plasmids of poIFNα1gene and three differentpoIFNα1-THYα1fusion genes were constructed successfully.The expression plasmids of poIFNα1gene and three different poIFNα1-THYα1 fusion genes were transformed into BL21(DE3) respectively, after induction withIPTG and ultrasonication, SDS-PAGE and Western blot results indicated that thesoluble poIFNα1protein and three different poIFNα1-THYα1fusion proteins wereexpressed in the E. coli cells successfully. In order to optimize the expression processof objective protein,9parallel experiments were designed and carried out byorthogonal experiments, after analysis of gray, the results indicated that the objectiveprotein reached its highest expression level （P<0.001）and constituted about22.13%of the total bacteria protein at30℃after induction with0.5mmol/L IPTG for12h.Accroding to the data analysis, induction time had the greatest effect on theexpression of objective protein, temperature came to the second and IPTGconcentration had the least effect.Drug standard has strict requirements for the purity of protein drugs, in order tomeet the requirements, ion exchange chromatography and hydrophobicchromatography were used to purify the poIFNα1protein and three differentpoIFNα1-THYα1fusion proteins, the purity was about90%. In order to identify theeffectiveness of ARG, the poIFNα1-LinkerA-THYα1and poIFNα1-LinkerB-THYα1fusion proteins were cleaved in vitro by thrombin and the cleavage succeeded at theARG site. In order to analyze the bioactivities of poIFNα1protein and three differentpoIFNα1-THYα1fusion proteins, cytopathic inhibition assay and erythrocyte rosettetest were used to analyze the IFN bioactivities of the poIFNα1protein and threedifferent poIFNα1-THYα1fusion proteins and the THY bioactivities of three differentpoIFNα1-THYα1fusion proteins respectively. The results indicated that the poIFNα1protein and three different poIFNα1-THYα1fusion proteins showed moderate IFNbioactivities, three different poIFNα1-THYα1fusion proteins also showed moderateTHY bioactivities, and the poIFNα1-LinkerB-THYα1fusion protein showed higherIFN bioactivities than others（P<0.001）, the poIFNα1-LinkerA-THYα1fusion proteinshowed higher THY bioactivities than others（P<0.05）.In this research, the poIFNα1-THYα1fusion proteins were expressedsuccessfully and the fusion proteins got a high purity and moderate IFN and THYbioactivities, but the purity and bioactivities have not met the requirements of drugstandard, so further optimization of purification process and improvement ofbioactivities will be made in the future, subsequently, the antiviral effects and other biological functions of poIFNα1-THYα1fusion proteins will also be analyzed in vivo,which will make a great contribution to the mass production and clinical applicationof poIFNα1-THYα1fusion proteins.