Studies On Prokaryotic Expression And Biological Activity Of Tianfu Rou Duck Interferon α

In this paper, the study is on Tianfu Rou Duck interferon. We conducted the following series of studies:Tianfu Duck typeⅠinterferon IFN-αORF gene sequence was cloned,identified and analysis; IFN-αORF prokaryotic gene expression vector was successful constructed;IFN-αhas been study by prokaryotic expression &.biological activity; The inclusion body was refolded by the methods of dilution, then the activity of the recombinant was detected by anti VSV in DEF. The results were as follows:1. Tianfu Duck IFN-αORF gene cloning, sequencing, analysis and protein structure prediction.Using software Oligo6.0, one pair of specific primers was designed within EcoRI and HindⅢrestriction sites.The total RNA was isolated from duck PBMCs stimulated with 5μg/ml PHA at 24 hours and transcribed to cDNA with oligodT,The expression of duck inteferon-α(DuIFN-α) was detected by reverse transcription-polymerase chain reaction (RT-PCR) method, a DNA fragment about 600bp(IFN-α) was amplified.and then this fragment was cloned into pMD18-T vector. The result of sequencing shows that the open reading frame(ORF) of DuIFN-αgene was consist of 576bp,and encodes a protein of 191 amino acids.The 30 amino acids at the N-terminus may serve as signal peptide.Mature polypetide of DuIFN-αseems to consist of 161 amino acids.Using DNAStar7.0 analysis software, sequence comparison with the published gooses IFN-αgene in GenBank (NO.AY524422), chickens IFN-αgene in GenBank (NO. AM049251), duck IFN-αgene in GenBank (NO.X84764), and other sequences in the GenBank. The results showed that Tianfu Rou Duck IFN-αsequences are very similar with duck(NO.X84764), its nucleotide homology to 99.5%, amino acid homology of 98.4%.And chicken nucleotide and amino acid homology are 73.4% and 51.3%, the goose IFN-αnucleotide and amino acid homology are 96.2%. and 92.15%. At the same time the gene protein was analyzed. The results show that Tianfu Rou Duck IFN-αhas two potential glycosylation sites, eight potential phosphorylation sites, and one transmembrane region.2. Tianfu Rou Duck IFN-αMature polypetide gene prokaryotic expression vector Construction, expression and polyclonal antibody preparationThe DuIFN-αgene was cloned specially into pET-32a(+) vector, and then the prokaryotic expression vector pET-32a(+)-mDuIFNαplasmid was constructedⅱrecombinant plasmid was transformed into prokaryotic expression strain E.coli (?) (DE3)pLysS. The recombinant fusion protein were expressed under IPTG induction,a polypeptide with a size corresponding approximately to that expected for the recombinant protein (about 35.0kDa) was highly expressed with 0.2 mM IPTG at 37℃for 4h,and found in large amounts in crude cell extracts. Purified protein was used to immunize rabbit for the duck IFN-αmature polypetide anti-serum preparation. Its agar diffusion reaction antibody titer was up to 1:32. High specific IgG polyclonal antibody was obtained by purification using the ammonium sulfate precipitation and High-Q anion-exchange chromatography.3.Refolding and antiviral activity detection on Tianfu Rou Duck IFN-αmature polypetideWe purified recombinant DuIFN-α,and refolding recombinant DuIFN-αwith dilution method, then the activity of the recombinant DuIFN-αwas detected by anti VSV in DEF. The results of antiviral activity are about 256 U/ml, vitality about 656.4U/mg. The results showed that the interferon has a strong biological activity in resistance to virus amplify.