Construction And Immune Effect Study Of Newcastle Disease–H9N2 Avian Influenza Chimeric Virus-like Particles

Both Newcastle disease and avian influenza are recognized as the two most important fowl blight in the world.Newcastle disease(ND)is an acute,heat,septic and highly contagious disease that is caused by Newcastle disease virus(NDV).It can be transmitted through many ways such as respiratory tract,digestive tract,eye conjunctiva and so on.The mortality rate is very high.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,H9N2 subtype avian influenza(AI)is a contagious disease caused by the H9N2 avian influenza virus(AIV).The transmission route is the same as NDV.H9N2 AIV is a low virulence strain,that in most cases is not fatal,but it will cause a serious decline in production performance of animal,poultry industry has been caused tremendous economic losses because of it.And because of the antigen drift and antigen conversion of the A influenza virus gene,AIV has caused great potential danger for public health safety.In clinical cases,NDV is often associated with H9N2 AIV,so it is necessary to have a joint prevention which both control of ND and H9N2.Mucosal membrane is the largest defense barrier of animal body,and it is the first defense line to resist the invasion of the pathogen.More than 95% of the pathogenic invasion occurred in the mucosal tissue.The data show that nasal immunity can not only induce good local immune protection,but also induce humoral immunity immune response.In many mucosal immune cells,Dendritic cells(DCs),as a professional immune cell,has the strongest antigen presenting ability,and dendritic cells participate in determining the nature,intensity and immune memory of immune response.The dendritic cell-banding peptide(DCpep)is a short peptide chain composed of 12 amino acids,which can specifically target DCs.It is beneficial for DCs to capture and recognize the antigen effectively,thus inducing the efficient mucosal immune response,and leading and regulating the mucosal immunity.It has been demonstrated that the recombinant antigen hepatitis C virus fused with DCpep(dendritic cell banding peptides)can effect DCs,induce the antigen-specific immune response,and found that antigen fused with DCpep did not modify DCs,the phenotype and function of DCs did not change,but mediated DCs targeting antigen to act a role as a kind of biological adjuvant effect.Virus like particles are highly structured hollow protein particles with no viral genome.Therefore,the virus-like particles has good imunogenicity and has no risk of spreading the virus genes.HN protein is the main immune protective antigen of NDV,and the specific antibody produced by the mouse is sufficient to protect the mice from the threat of NDV.HA protein is the major immune protective antigen of H9N2 subtype AIV.It can induce the body to produce effective neutralizing antibodies,which play a strong role in resistance the process of infection and pathogenesis.The research of Fan has shown that AIV HA expressed by recombinant baculovirus can induce strong immune response in mice,and can completely protect mice from H9N2 infection.To solve above problems,we constructed two new types of Newcastle disease avian influenza vaccines: NDV-AIV virus-like particles,which match with the popular strains and no gene pollution risk,and efficiently stimulated mucosal immunity protect animals from two disease with one operation.i)the extracellular domain of the F protein of NDV was replaced by the extracellular domain of the H9N2 subtype avian influenza virus HA,the chimeric gen named FcHA.ii)The DCpep gene is fused with the N end of the HA extracellular domain of the H9N2 gene and the fusion gene is used to replace the extracellular domain of the NDV F protein,the chimeric gen named DCpep-FcHA.The two groups were co expressed with HN on the surface of ND VLPs made up of M protein.They are named VLP-HA/HN and DCpep-VLP-HA/HN,respectively.The virus like particles were purified and immunized to SPF chickens in different ways.The immunogenicity of two virus-like particles and inactivated ND-H9N2 combined vaccine was compared.Combined with immunity,immune path,evaluated the better one of the two virus-like particles candidate vaccines,to provide a new strategy for the prevention and control of avian influenza and Newcastle disease.I.The construction and identification of recombinant baculovirus rBV-HA and rBV-DCpep-HA.First,primers were used to amplify the intracellular domain and transmembrane domain of NDV F and the gene extracellular domain of HA,and insert the enzyme cut site,and splicing them to construct FcHA by overlapPCR.DCpep-FcHA is kept by our lab,insert the enzyme cut site by PCR.Insert the target gene FcHA and DCpep-FcHA into the pFastBac 1 vector.Recombinant plasmid was sequenced and transformed into DH10 Bac,Referring to the Bac to Bac instructions,we extracted recombinant rBacmid-FcHA and rBacmid-DCpep-FcHA,then transfected them into Sf9 cells,obtained rBV-HA and rBV-DCpep-HA from culture supernatant,and identified the expression protein by Western Blot.II.Expression and identification of chimeric virus like particles VLP-HA/HN and DCpep-VLP-HA/HN.The baculovirus expression system was used to construct recombinant baculovirus rBV-HA and rBV-DCpep-HA,rBV-HA and rBV-DCpep-HA were cotransfected with the existing rBV-M and rBV-HN in the laboratory to cotransfect Sf9 cells,respectively,to express VLP-HA/HN and DCpep-VLP-HA/HN.The recombinant virus expressed VLP-HA/HN and DCpep-VLP-HA/HN.The protein expressed was identified by Western Blot.The virus-like particles with a diameter of about 80nm-200 nm were observed under scanning electron microscope.III.Evaluation of immune effect and effect of attack and protection of recombinant virus like particles VLP-HA/HN and DCpep-VLP-HA/HN.We use the method of intramuscular injection to immunize the SPF chicken with commercialized NDV-H9N2 vaccine.We immunized VLP-HA/HN and DCpep-VLP-HA/HN groups by intramuscular injection and intranasal instillation,after immunization,we test humoral immunity,cell immunity,and respiratory mucosal immunoassay.After immunization,the animals were attacked and the immune protection was evaluated by the aspects of weight,survival rate and virus distribution.Through the above experiments,we get the following conclusions.i)The two virus like particles constructed in this experiment have complete virus biological morphology and good antigenic biological activity.ii)Chimeric virus like particles can induce good humoral immunity,cellular immunity and mucosal immunity.The immune effect of DCpep-VLP-HA/HN was better than VLP-HA/HN and commercial vaccine.iii)the virus like particles can be used by no adjuvant,so they can immune through intranasal instillation,mucosal immune effect produced by intranasal instillation is better than intramuscular injection.iv)Compared with the traditional vaccine,DCpep-VLP-HA/HN has a simple immunization way,reducing the risk of dispersal,and shortening the time of virus in the chicken.It can show the advantage of the same genotype between vaccine and virulent strain.