Tianfu Goose IFN-α Gene Prokaryotic Expression And The Study Of Its Antiviral Biological Antivity

This scientific dissertation concentrates on studies on the identified Tianfu goose IFN-a (TFGoIFN-a) gene (Genbank accession no. HQ115583). The sequence characterization and codon usage bias analysis, cloning, prokaryotic expression, preparation of polyclonal antibody, renaturation of protein and the antiviral biological activity. Results were shown as following:1. TFGoIFN-a gene clone and sequence characterization analysisInduced peripheral blood mononuclear cell (PBMC) by CoA, extracted RNA and amplified TFGoIFN-a gene by RT-PCR, pMD19-TFGoIFN-a was constructed and sequenced. The ORF of TFGoIFN-a gene was 576 nucleotides encoding for a polypeptide of 191 amino acid residues with 21kDa. Furthermore, it was forecasted that signal peptide was composed by approximately 28 amino acid, the 28th and 29th amino acids for the cutting site of signal peptide and mature. TFGoIFN-a had a high degree of preference of G and C ending codons, and C3s (the third position of codon is C) content was 81.29%. Comparison of the codon usage in the TFGoIFN-a gene of different organisms revealed that there were 47 codons showing distinct codon usage differences between the TFGoIFN-a and Escherichia coli TFGoIFN-a genes; 46, between TFGoIFN-a and yeast dUTPase genes; and 45, between the TFGoIFN-a and human.2. Prokaryotic expression of pET-32-mTFGoIFN-a and polyclonal antibody preparationAccording to signal peptide position, a pair primers ware designed to amplified mature petide (mTFGoIFN-a) gene. Constructed pMD19-mTFGoIFN-a and sequenced, prokaryotic expression vector pET-32a(+)-mIFGoIFN-a plasmid was constructed after double digestion. A polypeptide with a size corresponding approximately to that expected for the recombinant protein (about 38kDa) were highly expressed with 0.8mmol/L IPTG at 30℃, and found in large amounts as inclusion body. Purified protein was used to immunize rabbit for the TFGoIFN-a recombinant protein anti-serum preparation. The potency of the anti-serum was 1:16 by ager diffusion test. It was shown a specific stripe by western bloting.3. Renaturation of recombinant protein expression and antiviral active detectionAfter dilution, dialysis and Ni-IMAC affinity chromatography, the protein was detected the anticities of recombinant protein was 104U/mL and specific activites was 5.5 X 103U/mg. It had the same results for GEF system. The test of anti-GPV, it was significant difference existed between virus copy number of the IFN protection group and GPV group (P<0.05), TFGoIFN-a recombinant protein has the antiviral biological antivity against GPV. The relative inhibition rate as high as 89.4%.