| Interferons (interferons, IFNs) are a multigene family of inducible cytokines which mediate antiviral, immunomodulatory, and antiproliferative effects. IFNs have broad applied prospects as biological agents and vaccine asjuvants.In order to discuss the possibility of recombinant goose interferon-a, in this study, the signal peptide gene of goIFN-a was removed and then was placed into the pichia pastorisα-factor secretion signal sequence to reconstruct the recombinant expression plasmid pPICZaA-goIFN-a. Then the recombinant plasmid was transformed into pichia pastoris host strain X33, and realized its high efficient expression. And its antiviral activity was evaluated by using VSV on goose embryo fibroblast, which provides a good foundation for the research and development of goose interferon. The main results of this reseatrch are as followed:1. The primers were designed and synthesized by the gene sequence of TianFun goose IFN-a, which had been submitted into NCBI (HQ115583). Then the goose IFN-a gene of mature peptide was amplified from the recombinant plasmid pMD 18-T-goIFN-a containing the complete sequence of Tianfu goose IFN-a by PCR, afer the PCR product was purified and recycled, it was colned into pMD18-T vector. After DNA sequencing was correct, the recombinant plasmids pMDT-IFNa was digested by EcoR I and Xba I, and ligated with pichia pastoris shuttle vector pPICZaA which was also digested by EcoR I and Xba I. Then the ligated liquid was transformed into E.coli DH5a to construct the recombinant expression plasmids pPICZaA-goIFN-a. The recombinant plasmids were suffered by PCR, restrict enzyme digestion and sequence analysis.2.The correct recombinant expression plasmid was digested by sac I and the linearized recombinant plasmid was transformed into pichia pichia strain X33 by electroporation. Putting them on the selective YPDS culture medium containing zeocin at 30℃for 3-5 days, and then many white transformants would been screened on the culture medium. Three different white single clonies was pick up and induced first by BMGY culture medium, and then using BMMY culture medium to resuspended the cell. After the culture medium was induced by adding 1% methanol every 24h for 72h, the culture was centrifugated and collected. The multi-copy recombinants were picked by dot blot hybridization, and then it was reinduced to express the protein, and then the expression conditions of the best time and methanol concentration were optimized by Elisa. After the recombinants were expressed by the best methods, it was concentrated by TCA and was deteced by SDS-PAGE and Western-blotting assays. 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.3. The antiviral activity was evaluated by using VSV on goose embryo fibroblast. The goose embryo fibroblast cells were preparated, and its half amount of infected with VSV was determinated by TCID50.Then after the protein was dialyzed and filtration sterilized, its antiviral activity was evaluated, and the rusults revealed that goose interferon-a could inhibit the VSV on goose embryo fibroblast and had an antiviral activity of 1.79×103U/ml.In summary, this study has successfully cloned goose interferon-a gene, and successfully constructed the eukaryotic expression plasmid of goIFN-a, achieved its highly expressed in pichia pastoris and the expression products have significant biological activity. |
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