Large-scale Fermentation Of Mature Chicken Interleukin-18Protein And Study On Its Immunoenhancement On Newcastle Disease Vaccine

Chicken interleukin-18(ChIL-18) was founded in2000, which has multiple biologicalactivities including induction of IFN-γ from NK cells and antigen-or mitogen-stimulated Th1cells, upregulation of IL-2R on T cells, enhancement of Fas ligand-mediated cytotoxicity ofT-helper cells and augmentation of NK cell cytotoxicity. The Pichia pastoris is one of themost successful freign protein expression systems until now. It offers economy, ease ofmanipulation, the aility to perform complex post-translational modifications, and highexpression levels. So it is important to direct the expression of a bioactive mature chickeninterleukin-18(mChIL-18) in P.pastoris. The objective of this research was to explore theprocess of large-scale mChIL-18fermentation expression with5L fermenter. It emphasizesthe factors significantly influencing the fermentation. mChIL-18liposomes can improved thebioactivity. Then explore mChIL-18immunoenhancement on Newcastle disease vaccine,which provides a good foundation for the research and development of mChIL-18. Mainresults of this research are as followed:The train GS115/pPIC9K-mChIL-18was inoculated in YPD medium and theninoculated into the fermenter until the OD600of the culture reached about5.2. Usingfed-batch fermentation, the high-density fermentation of genetically engineered Pichiapastoris was processed in a5L bioreactor. After induction for84h, the target protein waspurified by6×His-Tagged Protein Purification Kit. The optimum expression conditions asfollows: temperature28℃, pH5.5, the DO20%-30%and the concentration reached560mg/L. After the purification, the purity could be more than70%.According to a certain proportion of using mChIL-18, cholesterol and phosphoric tomake mChIL-18liposomes and the encapsulation rate was25.4%.And, to study the immunity enhancement of IL-18, one-week-old SPF chickens wererandomly divided into4groups. Group I received vaccine. Group II received0.2mLmChIL-18liposomes and vaccine. Group III received liposomes and vaccine. Group IVserved as0.9%Nacl control and the normal control. The results were analyzed at the level of humoral and cellular immunity to interpret the immunity enhancement. The results showedthat the level of HI antibody deteceted in group II was always higher than other groups atdays14post-immunization. It presented significant difference (P<0.05) between group II andgroup I at days14,21and28post-immunization. The level of cellular immunity wasdetected by FACS. The number of CD4~+and CD8~+T lympholeukocytes in peripheral bloodof chickens immunized with genetically engineered mChIL-18liposomes were obviouslyhigher than group I. Group IV was always higher than group III and group I, and thenpresented significant difference. The number of CD4~+and CD8~+T lympholeukocytes inmChIL-18liposomes immunized group kept increasing tendency and longer persistencecomparing with others group after the secondary immunization. It presented significantdifference (P<0.05) between group II and other groups. Attacking poison experiment showedthat the group of simultaneous inoculation of mChIL-18liposomes and vaccine gains86.7%conservation ratio while the vaccine class gains66.7%.Thus, as immunity adjuvant, IL-18plays an important role in enhancing immunity andimmunity effects.