Expression Of Porcine Interferon Gamma Gene In Pichia Pastoris And Determining Of Anti-vsv Activity

Porcine interferon-gamma (PoIFN-γ),produced by activated cytotoxic T cells and natural killer cells, plays important roles on anti-infectious, Antitumor Activity and immunomoduLatory effects.PoIFN-γhave capacious applied foreground as biological fungicide and Vaccine Adjuvant.In order to develop the application of recombinant PoIFN-γ, a recombinant expression vector was constructed in this study.The porcine interferon-gamma gene, in which the sequence encoding signal peptide was cutted then followed that of the a-factor of Saccharomyces cerevisiae, was cloned into Pichia pastoris expression vector pPIC9K. in order to secrete express the PoIFN-γ.The recombinant plasmid pPIC9K-PoIFN-γwas constructed. The objective of this research is to transform the cloned PoIFN-γgene into Pichia pastoris. Then culture the Pichia pastori, expressing the PoIFN-γ, determining the activity. which provides a good foundation for the research and development of PoIFN-γ.Main resuLts of this research are as followed:1,Expression fragment of mature protein of PoIFN-γabout 441bp was obtained from recombinant plasmid of PMD-18T-PoIFN-γcontaining complete PoIFN-γby PCR amplification with primers designed according to the sequence PoIFN-γ. Desired fragments obtained from double-digestion of plasmid PoIFN-γand pPIC9K with EcoRI and NotI were restored and linked together. thus a recombinant plasmid vector pPIC9K-PoIFN-γwas constructed. Sequencing resuLts of plasmid pPIC9K-PoIFN-γindicated that structural PoIFN-γgene was integrated into the correct reading frame.2,salⅠ-linized recombinant plasmid pPIC9K-PoIFN-γwas transformed into Pichia pastoris by produce plasmatosome.53 positive transformants were screened on MD plates containing G418. Spcific Pichia clony PCR product showed that foreign PoIFN-γgene was integrated into the host cell. The experimental resuLts from SDS-PAGE and protien of PoIFN-γactivity assay showed recombined pichia was successfuLly transformed into by the PoIFN-γgene.3,combined pichia cell was cuLtured in the MGY.The experimental resuLts of SDS-PAGE assay of cuLture medium showed protein was not successfuLly secreted expression. But the 17KD target proteins can be found in the uLtrasonic lysate. The specific activity of uLtrasonic lysate was assayed by the standard atuviral activity test on WISH cells challenged with VSV virus.resuLt showed.The recombinant PoIFN-γcan well protect WISH cells against viruLent vesicuLar stomatitis virus (VSV) infection in vitro.