Expression Of Chicken Interleukin-2 In Pichia Methanolica And Analysis Of Biological Activity

Domestic infective diseases wich caused the tremendous economic losses to poultry industry, were the most large threat of poultry industry. The application of vaccines was the most improtant strategy to guard against these diseases. In the poultry industry, there are various vaccines to different diseases, but they are not effective to all diseases. In addition, vaccines could not entirely protect fowls from given disease. In particular, the low antigenicity of inactivated vaccines and genetically engineered vaccines often cause failure during immunization.Chicken interleukin-2 (ChIL-2) can be used as immunological adjuvant in the poultry industry to enhance the immunity of chickens against pathogens. It had been a focus in the cytokines'study since it was found in 1982. It was used as an important adjuvant in cell immunity.In the present study, the chicken interleukin-2 gene was inserted into the expression vector pPIC9K to construct recombinant plasmid (pPIC9K/ChIL-2) followed by linearizing by restriction endonuclease Sal I. Then it was transformed into host Pichia Pastoris GS115 by eletroporation. After screening by G418-YPD, following by MM and MD plates, we obtained 5 positve clonies (His+Mut+). In the next PCR, 3 clonies with high-copy of purpose gene were obtained. Among them, one colony was cultured in BMGY/BMMY flask for the expression of ChIL-2 in vitro. The expression of recombinant ChIL-2 was successfully performed. Similarly to native ChIL-2, the expressed ChIL-2 revealed two main bands corresponding to molecular masses of 16 and 14 kDa, respectively as detected by SDS-PAGE and Western blot, suggesting it could be a glycosylated protein.By fermentation, the production of recombinant protein increased about 20-fold, up to 100 mg/L. In the total cell proteins, the target protein ChIL-2 occupied more than 50 %. Because the great difference of molecular masses between the proteins in the cell culture, membrane ultrafiltration purification was applied to ChIL-2 purification and was a convenient way. The present study established a foundation for the industrial production of ChIL-2.Purified ChIL-2 was co-admitted with avian influenza virus (AI) vaccine and newcastle disease (ND) vaccine to study its immunological enhancing effect on AI and ND in chickens.The antibody titers of AI and ND were detected by hemagglutination inhibition test. The results indicated that ChIL-2 had positive immuno-enhancing effect on both vaccines. The immuno-enhancing effects were respresented by antibody titer increasing and its extending. ChIL-2 also increased the survival rate of the chickens. The optimum dosage of ChIL-2 as adjuvant was about 8.35μg/chicken.During the whole experiment periods,no toxic effect of ChIL-2 was identified, suggesting it was an effective and safe adjuvant for vaccination.