The Clone Of PoIFN-a And Its Expression In Escherichia Coli

Interferons (IFNs), a family of cytokines, have many kinds of biological activities, such as interfering with the replication of various viruses, decreasing cell proliferation and modifying immunological processes. IFN- a / P is the first system of body to defend virus attacking. Pig production is important for food industry and the development of recombant porcine interferon- a ( rPoIFN- a ) is strongly desirable for prevention of pig virus diseases.A pseudogene with a termination codon TAG in the middle was isolated from porcine liver by PCR method. The sequence had been send to Genebank and the accession number was AF350425. Then a PoIFN-a gene was produced by AS-PCR and SOE using four special primers based on the differences between the sequence of the pseudogene and PoIFN- Q 1. hi the sequence of the PoIFN- a gene five nucleotides were different from the sequence of PoIFN-a i, which leaded to three amino acids difference.To obtain a recombinant mature PoIFN- a , plasmid pGRW-PoIFN- a 1 a was constructed. The expression of the unfused protein in the strain E. coli DH5 a was 10%, and the antiviral activity of the protein was 3200IU/mg. Then a site-directed mutation of Cys(86) to Tyr was made by megaprimer PCR ; meanwhile TGT, the first codon of PoIFN- a , was changed to TGC , which was a bias codon to E. coli. In order to increase the expression level of PoIFN- a , the mutated sequence was inserted into the fused protein expression plasmid pGEX-4T-3. The expressed fused protein, which was found in insoluble form, counted for 20% of the total cell proteins. After denaruration, renaturation and purification, a higher antiviral activity was measured (5200IU/mg).