Expression Of Newcastle Disease Virus HN Protein In Pichia Pastoris And Study Of Its Immunogenicity

Newcastle Disease is an acute and highly contact infectious disease of poultry caused by Newcastle disease virus(NDV).Its main clinical manifestations are serous mucosal hemorrhage,dyspnea,chin,and neurological disorders.The disease spreads rapidly,has a high mortality rate,is highly contagious and widely distributed,and has had a major impact on the global poultry industry.Although attenuated and inactivated vaccination of poultry is the most effective way to prevent and control Newcastle disease,the continued outbreak of Newcastle disease in commercial poultry flocks worldwide has shown that routine vaccination is not enough to control the disease.Therefore,there is a requirement to develop more secure and reliable Newcastle disease vaccines.HN protein is the main structural protein of Newcastle disease virus and the main target of the NDV immune response.It has good immunogenicity and can induce immune response.It has always been a research hotspot of Newcastle disease subunit vaccines.The Pichia pastoris expression system has significant advantages such as fast growth rate,post-translational modification,secretory expression,and easy genetic manipulation,and is an ideal foreign protein expression system.In this study,we chose Pichia pastoris expression system to express HN protein,and tested the immunogenicity of HN protein with SPF chickens as experimental animals..The main work is as follows:1.Expression and identification of HN recombinant proteinIn order to obtain the immunogenic HN protein,the optimized extracellular domain HN gene was cloned into pPICZaA,and the pPICZaA-HN was electroporated into Pichia pastoris X-33 competent cell.The positive transformants were obtained by PCR identification.The positive pichia pastoris was induced by methanol,then identified by SDS-PAGE and Western-blot.The results showed that HN recombinant protein was successfully expressed in pichia pastoris.The optimal expression conditions were explored by optimizing the methanol concentration,induction time,initial pH of the medium,and induction temperature.The results of Dot-blot showed that at 28?,the initial pH of the medium was 7.0,and the expression of HN protein was the highest after 5 days of induction with 0.5%methanol.2.Purification and activity identification of HN recombinant proteinThe HN recombinant protein was purified by Ni column affinity chromatography,and the results of SDS-PAGE showed that the purity of HN recombinant protein purified by Ni column could reach 90%,and its expression was 25 mg/L by BCA kit.The results of indirect ELISA showed that the HN recombinant protein could specifically bind to NDV positive serum,indicating its good activity.Deglycosylation assays demonstrated the presence of glycosylation modifications in HN recombinant proteins.3.Evaluation of the immune effect of HN recombinant proteinThe purified HN recombinant protein was emulsified with Freund's adjuvant and immunized with SPF chicken.After immunization,the titers of NDV antibody and hemagglutination inhibition antibody in serum were detected weekly,and the highest antibody titers reached 212.4 and 26.2 respectively.The results indicate that the HN recombinant protein stimulated a high level of humoral immune response in SPF chickens.In addition,the levels of IL-2 and IFN-y in the serum of chickens on the 21st day after second immunizations were tested.The results show that the HN recombinant protein can induce cellular immune responses.The immunized chickens were challenged at 21 days after the second immunization.The results showed that the immune protection rate of the HN protein-immunized group was 70%,and can reduce the damage of tissues and organs,and has the potential to become an NDV subunit vaccine.In summary,this study successfully established a high-efficiency Pichia pastoris expression system for NDV's HN recombinant protein,and obtained high-purity and highly active HN recombinant protein.Recombinant HN protein can protect 70%of SPF chickens from lethal attacks.It laid the foundation for the development of Newcastle disease subunit vaccines and detection reagents.