Cloning, Expression And Biological Activities Of The Tianfu Goose Interferon-alpha

China is an important country of waterfowl breeding. Tianfu Rou goose with good hereditary capacity, is widely breeding in Southwest China, and brings significant social and economic benefit. With the development of intensive aquiculture of goose bleeding, it's significative to research biological agent to enhance immunity for goose vaccine and to take effect on virus. IFN-a has bioactivity of broad antiviral and immunoregulation. The subject is to study clone, expression, protein function and application base on the technology of genetic engineering.1.Cloing and sequencing analysis of the tianfu goose IFN-αThe tianfu goose interferon-alpha (tf-GoIFN-a) was amplified from peripheral blood mononuclear cells by RT-PCR, and then it was cloned into the vector of pMD18-T to construct the recombinant plasmid. The nucleic acid and amino acid sequence of (tf-GoIFN-αwas analyzed with bioinformatics software and on-line tools. The similarity and evolution with 13 other correlative gene sequence was also analyzed. The results revealed that the tf-GoIFN-a was cloned successfully, the whole ORF of the tf-GoIFN-αcontained 576bp nucleotides, encoded a polypeptide of 191 amino acid residues with a predicted molecular mass of 21670.7 kDa. The possible potential cleavage site is between the 28th (Alanine) and 29th (Phenylalanine) amino acid residues. Its secondary structure was composed by Alpha helix and Random coil, mainly including ten antigenic determinants. The result from analyzing the tf-GoIFN-αamino acid sequence reveals that there is two N-glycosylation sites, nine O-glycosylation sites, five potential phosphorylation sites,the first hydrophobic region is the same as the signal peptide, meaning the signal peptide would guide the protein to secret into the exrracellular, there is no transmembrane domain. The analysis of the protein subcellular location indicates that the tf-GoIFN-a locates at extracellular with 55.6%, cytoplasmic with 22.2%, mitochondrial with 11.1% and nucleus with 11.1%. Similarity comparison shows that the tf-GoIFN-a has more than 90% similarity to waterfowl (duck and goose) and in the same branch in the phylogenetic tree. It has the most similar with the Shitou goose, of which 99.3% nucleotide similarity and 97.9% amino acid similarity. The codon analysis showed that the tf-GoIFN-a gene which we have cloned was high expressed and high codon bias gene. Its codon usage situation is similar to duck IFN-a, but is different from chicken IFN-a, which means that we can express the tf-GoIFN-a as the duck IFN-a and then make the future sudy.2.tf-GoIFN-a mature polypetide gene prokaryotic expression and antiviral active detectionBased on the cloned ORF gene of tf-GoIFN-a, one pair specificity primer was designed to amplify the mature peptide. It was obtained a gene of the length of 483bp encoding 161 amino acids. Then the mtf-GoIFN-a gene was cloned into pET-32a(+) vector specially, and prokaryotic expression vector pET-32a(+)-mtf-GoIFNa plasmid was constructed. Transforming into prokaryotic expression strain Rosetta, a protein of 38kDa was expressed. After renaturation by Ni-IMAC affinity chromatography, dilution and dialysis, it was shown one specificity strip. Antiviral activity of IFN was examined by assayed by cytopathic inhibition assay on goose embryo fibroblasts. The anti-VSV activity of renaturation protein was 104U/mL,and specific activity was 5.5×103U/mg. And the detection result of anti-VSV activity was consistency in GEF and DEF. It was detected anti-GPV activity of tf-GoIFN-αprotein by SYBR Green real-time PCR. The result showed that there was significant difference (P<0.05) between the IFN protection group and GPV group. In other words, tf-GoIFN-a protein has the activity of anti-GPV. And the relative inhibition rate to GPV was 89.4%.3. Purification of goose IgG and preparation of rabbit anti-goose IgG-HRPTo study the preparation of rabbit anti-goose IgG HRP antibody,test employed the method of diluted with water--n-octylic acid--ammonia sulfate initial extraction, out salt dialysis and DEAE chromatography, detected the IgG concentration and purity of initial extraction by protein nucleic acid detector and SDS-PAGE, prepared rabbit anti-goose IgG HRP. Results showed:crude goose egg IgG had high concentration (10mg/mL) and purity(94.5%), the titer of the immunodiffusion (1:32), the titer of HRP labeled Antibody(1:3000).4. Construction of a recombinant eukaryotic expression plasmid containing tf-GoIFN-αgene and its transient expression in COS-7 cells To further study the tf-GoIFNa gene character and functions, the gene tf-GoIFNa was cloned into eukaryotic expression vector pcDNA3.1-(+) to generate the recombinant plasmid pcDNA3.1-GoIFN-a, which was then transfected into the COS-7 cells by lipofectin transfection. Then, its expression and localization in COS-7 cells were determined by indirect immunofluorescence, enzyme-linked immunosorbent assay and Western-blot assay, and the antiviral activity of supernatant from transfected COS-7 cells was also determined.The results indicated that eukaryotic expression vector pcDNA3.1-GoIFN-a was successfully constructed, tf-GoIFNa protein was fist detected at hour 12 post-transfection, increase was rapid in the supernatant before 36h, thereafter it became slow; Recombinant plasmid could be highly expressed in COS-7 cells, and a protein of 38 kDa in transfected COS-7 cells was also detected,which was larger than the predicted molecular weight of 21 kDa; Indirect immunofluorescence showed that tf-GoIFNa gene products diverged from the nucleus to the cytoplasm;The antiviral activity of supernatant against (VSV) and gosling plague virus (GPV) was obvious, as verified by method of cytopathic effect inhibition test.5. Adjuvant effect of eukaryotic expression plasmid containing tf-GoIFN-αgene on GPV-VP3 DNA vaccineIn order to study on the adjuvant effect of tf-GoIFNa eukaryotic expression plasmid to Goose Parvovirus VP3 gene vaccines, pcDNA-GoIFN-a with GPV-VP3 gene vaccines was inoculated to 28-day old geese at the dose of 50μg,100μg,200μg per goose respectively by intramuscular injection,the control groups included GIL2 molecular adjuvant,GoIFN-αand gIL2 combined immune adjuvant,empty vector plasmid pcDNA3.1 (+), Gosling Plague Attenuated vaccine and saline, anticoagulated blood samples were harvested at 3,7,14,21,28,35,49days to to detect the proliferation of geese T lymphocytes in peripheral blood(PBL) by T lymphocyte proliferation tests (MTT); and cruor samples harvested at 3,7,14,21,28,35,49,63,77 days to detect anti-GPV antibody by indirect ELISA methods and neutralization tests;obtaining the following experimental result:1). The reaction of T lymphocyts to ConA (OD value):The reaction of T lymphocyts to ConA (OD value):After different doses of pcDNA-GoIFN-a were administrated to geese with GPVVP3 DNA vaccine, the experimental groupsT lymphocyte proliferation were at the same level at 3d, from 3d to 28d the steady rise and at 28d reached a peak,but pcDNA-VP3 group peaked at 35 d.At 28d, the OD value of the adjuvant groups were significantly (P<0.01) higher than pcDNA-VP3 gene vaccine group, GPV attenuated group, pcDNA3.1 (+) and saline control group.GolFN-αadjuvant groups only at 21d were significantly (P≤0.05) higher than gIL2 adjuvant group, the other time pointed had no significant difference. United adjuvant group at]4d,28d to 49d was significantly higher than GoIFN-αor gIL2 adjuvant group (P≤0.05).2). The level of antibody IgG in PBL(OD):Adjuvant groups of ELISA antibody levels of the growth trend was from slow to fast, to reach the maximum value at 35d, then gradually decreased, but still significantly higher than GPV-VP3 gene vaccine group and GPV attenuated group.GIL2 adjuvant group at 28d to 42d,63d to 77d was significantly higher than GoIFN-αadjuvant group (P≤0.05), GoIFN-αand gIL2 combined adjuvant group at 28d to 49d was significantly higher than GoIFN-αor gIL2 adjuvant alone group (P< 0.01). GoIFN-a adjuvant groups, along with the different adjuvant doses showed a dose relation.3).Neutralizing antibody titer in PBL:At 60d and 75d, GoIFN-αand gIL2 combined adjuvant group was significantly (P<0.01) higher than GoIFN-αor gIL2 adjuvant group. In the adjuvant groups, the antibody titers in the first period of 45d to 75d were significantly (P≤0.05) or highly significant (P≤0.01) higher than that of pcDNA-VP3 group. GPV attenuated group was significantly higher than GoIFN-a or gIL2 GoIFN-a adjuvant group alone at 45d and the antibody levels induced (P≤0.05), was not significant with GoIFN-a and gIL2 United adjuvant group. The entire testing process, the injection of pcDNA3.1 (+) and saline control groups can not protect cells against Gosling plague virus infection.The results demonstrated that pcDNA-GoIFN-a could enhance the ability of T lymphocytes proliferation, enhance the titre of antibody IgG and neutralizing antibody in PBL, is an excellent GPV-VP3 gene vaccine molecular adjuvant,GoIFN-αand gIL2 combined adjuvant group had best effect, pcDNA-GoIFN-a intramuscularly 200μg was better.6. Secretory expression of tf-GoIFN-αin pichia pastoris and its bioactive researchIn order to make better use of genetic engineering techniques to develop recombinant goose IFN-a preparations. The goose IFN-a gene of mature peptide was amplified by PCR from the recombinant plasmid pMD18-T-GoIFN-a which included the whole ORF of tf-GoIFN-α, and colned into shutter vector pPlCZaA to construct the recombinant plasmid pPICZαA-GoIFN-α. The recombinant plasmids were linearized by sac I and transformed into pichia X33 cell. After three different strains were induced by 1% methanol for 72 hours, the multi-copy recombinants were picked by dot blot hybridization and the expression conditions of the best time and methanol concentration were optimized by Elisa. Afer the recombinants were expressed and concenrated by TCA, it was analyzed by SDS-PAGE and Western-blotting, and its antiviral activity was evaluated by cytopathic effect inhibition. The results revealed that the the pastoris expression plasmid of goose IFN-αwas constructed successfully, and achieved its highly expressed. The SDS-PAGE and Western-blotting assays demonstrated that tf-goIFNα, about a 22 kDa protein, was successfully secreted into the culture medium, it was a little bigger that the predicted (18.7 kDa), it was estimated that the expression products was glycosylated durning the expression. The expression products could inhibit the vesicular stomatitis virus on goose embryo fibroblast and had an antiviral activity of 1.79×103U/mL.