Construction And Immune Effect Study Of The Genotype ? Newcastle Disease Virus-like Particles

Newcastle disease(ND)is an acute,heat,septic and highly contagious disease that is caused by Newcastle disease virus(NDV).Vaccination is one of the most important measures to control ND.At present,gene analysis based on NDV-related molecular epidemiological data shows that the genotype VII has become a major dominant genotype against poultry industry in China and other countries,however,commercially available vaccine is the genotype II La Sota strain,which is proved to be antigen differences compared to the genotype of current circulating strains,thus,immune bias is caused.It suggests that there is undoubtedly a great advantage and potential if we use the genotype-matched vaccines to control ND.Virus-like particles(VLPs)are constructed by the viral structure protein to correctly simulate the natural virus,and assembled into a structure without any infectious and genetic material of the virus.Currently studies have shown that it has a great potential for prevention when used as a vaccine.Based on the baculovirus vector expression system and VLPs vaccine production platform,in this study,we used the genotype VII NDV NA-1 strain as the research object,then the genotype VII Newcastle disease virus-like particles with F and HN protein were simultaneously expressed with NDV M protein.In order to prepare the genotype VII Newcastle disease virus-like particles,a single cistron recombinant baculovirus respectively encoding the structural genes of Newcastle disease virus M,F and HN was constructed by using baculovirus vector expression system.Cytopathic effect of Spodoptera frugiperda 9(Sf9),the results of PCR and indirect immunofluorescence showed that recombinant baculovirus were successfully constructed after transinfection.The collected cell supernatants after co-infection were purified and concentrated by ultracentrifugation and sucrose density gradient.Then,the results of western bloting and transmission electron microscopy showed that the three target proteins were correctly expressed and assembled into particles whose configurationis similar to that of Newcastle disease virus and its size is in range of 200 nm.The titer of hemagglutination could reach 12log2 after the ultracentrifugation and sucrose density gradient purification.The results showed that the three target proteins were correctly assembled into ND VLPs with high hemagglutination activity,which laid a foundation for further study of their immunogenicity.In order to further study the immune effect of the genotype VII ND VLPs,in this study,commercial chickens was used to immunize with ND VLPs containing 25 ?g protein or combined adjuvant and the serum HI antibody were weekly monitored.The results showed that the ND VLPs or combined adjuvant could induce higher HI levels.The challenge assay showed that ND VLPs or combined adjuvant could provide 100% protection when resistant to the infection of the current epidemic strains.Compared with La Sota inactivated oil emulsion vaccine,ND VLPs or combined adjuvant effectively reduce virus shedding from oropharyngeal and cloacal,and virus load in chicken lung tissue,thereby reducing the chance of virus infection in healthy flocks.In this paper,ND VLPs formed by NDV M,F and HN were successfully constructed and expressed by baculovirus vector expression system.ND VLPs are capable of producing higher levels of HI antibodies,which could effectively reduce the virus sheding.The results showed that the genotype VII ND VLPs have a great advantage as vaccine candidate.