Prokaryotic Expression And Actvity Assay Of Recombinant Porcine Interferon-alpha

Porcine Interferon-alpha (poIFN-α) is a kind of cytokine with antivial activities, anti-tumor and regulatory immunity functions, especially its antivial activities. The study shows that the poIFN-αhas significant therapy at a variety of swine viral infect -ious diseases, such as epidemic diarrhea, swine plague, blue ear disease and infectious gastroenteritis. At present, the research on developing the Recombinant Porcine Interferon-alpha has become a hot spot, and a number of species and various types of interferon products hasbeen on sale, but it still far from being unable to meet market demand. In this paper, we expressed and purified recombinant poIFN-αwith biological activities successfully, and the recombinant poIFN-αwill be developed further.1,Rebuilding and cloning of the Porcine Interferon-alpha geneIn this study, we optimized the original sequence of poIFN-αaccording to the table of codon usage for E. coli highly expressed genes. Compared with the original sequence, the optimized sequence was changed 79 bases and 63 codons. Because there were five cysteine codons in the original sequence, we changed the number 86 cysteine codon (UGC) in the original sequence to the tyrosine codon for avoiding the mismatch of disulfide bond.The optimized sequence was divided into 12 parts, then, we spliceded the new sequence by the method of the SOE PCR (gene splicing by overlap extension PCR); The DNA fragement obtained was cloned into pGEM-T vector, the result of the restriction nucleases digestion and DNA sequencing analysis showed the cloning vector pMT-poIFN-αwas constructed successfully. 2,Prokaryotic expression,identification and purification of the recombinant recombinant porcine Interferon-alphaAccording to the optimized sequence, we designed the primers, amplified interferon gene using PCR, and then constructed the prokaryotic expression vector pET28-poIFN-α. Restriction analysis and sequencing proved that the prokaryotic expression vector pET28-poIFN-αwas successfully constructed. The pET28-poIFN-αwas induced with IPTG to express. The expressed product in a form of inclusion body showed a relative molecular mass of about 21 kDa, contained 39.8% of total somatic protein and reached a purity of more than 90% after purification.3,The anti-viral activity of the recombinant porcine Interferon-alphaWe used the way of dilution to renature the recombinant porcine IFN-αafter purification; The anti-viral activity of therecombinant porcine IFN-αwas identified with MDBK/VSV and PK15/VSV systems, and the result showed the recombinant porcine IFN-αhad good anti-viral activity. Next, the activity of the recombinant porcine IFN-αpurified was verified by inhibiting the cytopathic effect. As a result, the activity of recombinant porcine IFN-αagaist VSV at MDBK was up to2.19×106 U/mg. In conclusion, the recombinant protein with good biological activities has been expressed in E.coli successfully.