Research On Genetic Stability Of Recombinant Pichia Pastoris And Purification Strategy Of Poifn-a

The first part of the thesis described the investigation on genetic stability of the recombinant P.pastoris strains expressing PoIFN-a and their growth characteristics. The results showed the original strain(S strain) could grow a little better(OD600:19～25) than passaged strains (N strains) (OD600:10～22) although their expression of recombinant protein remained at the same level of 1g/1. The target protein expressed and inserted gene was identified with different generations of the recombinant P.pastoris, which confirmed the genetic stability of the recombinant P.pastoris strains. Different induction temperature resulted in different expression level and integrity of target proteins. Lower induction temperature (20℃) was beneficial to target protein expression and integrity. Higher induction temperature of 30℃could cause complete degradation and reduced expression of target protein.The second part of the thesis described the research on PoIFN-a purification strategy. Large proportion of PoIFN-a could be precipitated from the supernatant with PEG6000. The target protein could be re-dissolved and recovered. The recovery rate is about 80%. The process needed to be operated in low pH circumstance because a pH>7 could lead to un-dissolvable precipitation. The precipitation condition was designated temporarily as pH6, NaCl 0.2mol/l, PEG6000 O.lg/ml. higher degree purification conditions with gel filtration Sephadexl6/40 (Sephadex G-50, Fine) were explored and a recovery of 60% and purity of 90% were obtained with target protein.Antiviral activity of PoIFN-a reagent was determined with WISH-VSV system. The results showed an antiviral bioactivity of original fermentation is 1.28×105 IU/ml, specific activity is 2.46×104 IU/mg; purified fermentation with method of PEG6000 is 2.9×105 IU/ml, specific activity is 1.04×105 IU/mg; gel chromatography is 8×103 IU/ml, specific activity is 6.7×103IU/mg.