Expression And Large-scale Fermentation Of Porcine IFN-γ In Pichia Pastoris

The IFNs were originally discovered as agents that interfere with viral replication ,now, they are classified into typeⅠ, typeⅡa nd typeⅢaccording to receptor specificity and sequence homology . The typeⅠIFNs are comprised of multiple IFN-αsubtypes, IFN-β, IFN-ω, IFN-ε, IFN-κ, IFN-τand IFN-ζ. IFN-γis the sole typeⅡIFN . TypeⅢwere discovered in 2003, which includes IFN-λ1, IFN-λ2 and IFN-λ3 . The typeⅡIFN are also called immune interferon ,it is one of the most important cytokines in vivo . A number of researches have dedicated that aside from the anti-virus ability , IFN-γcan coordinate immune response through transcriptional regulation of immunologically relevant genes , which make IFN-γa research focus of many subject, such as the immunology, genetics ,and molecular biology . Now, human IFN-γhas been utilized in clinical treatment of many diseases.Animal infectious disease caused by bacteria, viruses and other pathogenic microorganisms not only restrict the development of natural and regional faming industries, but also affect human health . the multiple biological abilities entitle IFN-γthe potential to prevent and cure the disease . To apply the IFN-γto clinic , people has been exploring the production of IFN-γ, but failed to meet the need of clinic . With the development of genetic engineering , it has been possible to produce the high bioactivity IFN-γwith low cost in large scale with high efficiency and stable expression system , and this facilitate the clinical application of IFN-γin treatment of animal diseases and the application as immune adjuvant .It is helpful for IFN-γto achieve its social and economic benefits.The object of the study is poIFN-γ, to explore the conditions for secrete expression and large scale production of poIFN-γmature polypeptides gene in P.pastoris which is an efficiency expression system of foreign protein , and the purification of poIFN-γas well . This study laid the foundation for clinical application of poIFN-γ.To prepare the detective serum of poIFN-γexpressed in P.pastoris . poIFN-γgene was expressed in E. coli to prepare antigen for immunization . For this purpose , the prodaryotic expression vector pET-28a(+)-poIFN-γwas constructed , and the target protein was expressed in E.coli BL21 (λDE3) in the form of inclusion bodies , after being purified ,the inclusion bodies were used to immune mice to produce anti- poIFN-γserum. The Pichia pastoris expression vector pPIC9K- poIFN-γwas also constructed , and it was transform into Pichia pastoris GS115 via electroporation.,and screened a strain which expressed poIFN-γstably and effectively by G418 and induction.Culture the engineering bacteria in a gradually enlarge manner .In the process of training , explored the optimal conditions for the engineering bacteria to express poIFN-γ, and fermentd poIFN-γin 50L fermentator .The optimal condions were : BSM medium with initial glycerol concentration of 40g/L ; induded the culture with methanol after 22-24 training ; the pH value was 5.0 during the cell growth phase , and it should be adjust to 6.0 at the beginning of induction ; the dissolved oxygen maintained at 35%-45% , the concentration of poIFN-γin the broth reached 4000mg/L after being induced for 48h , the fermentation broth is centrifuged and the supernatant was concentrated by hollow fiber ultrafiltration . The target protein was purified by hydrophobic chromatography, gelfiltration chromatography and ion exchange chromatography , finally, the purity of poIFN-γwas more than 90% .Investigated the immunological and biological activity of poIFN-γexpressed both in Pichia pastoris. poIFN-γprepared in the study could bind anti- poIFN-γserum specifically ,which demonstrated the immunological activity of poIFN-γ.Investigated the biological activity of poIFN-γexpressed in Pichia pastoris with PK-15/PRV system ,and compare it with poIFN-γexpressed in E.coli .The result showed that the antiviral activity titer of expressed in Pichia pastoris was as high as 1.1×109U/mg, which higher than poIFN-γexpressed in E.coli. Another detection system PK-15/HCV was also involved in the study to investigate the biological activity of the product expressed in the two expression systems, and the poIFN-γproduced by Pichia pastoris also showed a higher antiviral activity than the poIFN-γproduced by E.coli . It is the first time in this study to produce poIFN-γwith high biological activity and purity in fermentation .It has laid a good foundation to apply poIFN-γto clinic.