Construction And Identification Of H9N2 Subtype Avian Influenza Virus-like Particles

Avian influenza(AI)is a major infectious disease of poultry industry in China,which remains the focus of the prevention and control of avian infectious diseases at present and in the future.Avian influenza virus(AIV)of H9N2 subtype is widely prevalent in poultry of China,and the infection of H9N2 virus may lead to different degrees of respiratory symptoms,decreased egg production about 10-20%,and declined poultry immunity.The infected chickens are vulnerable to secondary infections by many kinds of bacteria and viral diseases.H9N2 AIVs can spread across species,and even threaten human health and life.In addition,H9N2 AIVs has provided 6 internal genes for the prevalent H5 subtype and H7N9 subtype AIVs in China.The infectious of these novel viruses caused great public health concern.Therefore,it is significant to prevent and control AIVs of H9N2 subtype in poultry.At present,vaccination is the main means to prevent and control AI in China.AIVs has many subtypes and it evolve frequently in the natural environment,which brought great difficulties to vaccine research and development.Virus like particles(VLPs)is a new strategy for influenza vaccines.Instead of depending on the traditonal production system of chicken eggs,it produces antigens by cell culture method.VLPs has the similar natural conformation with the virion.VLPs is more secure since it does not contain the infectious viral genome.In this study,the H9N2 genotype of avian influenza virus subtype 57 was studied.And the strategy of HA-NA-M co-expression was adopted in this study to produce VLPs.Firstly,we constructed the recombinant transfer vector which can let to HA-NA-M1 co-expresssion.The M1 gene was inserted downstream of promoter pH of the p FastBac Dual vector,and the HA and NA gene was inserted downstream of promoter p10 of the vector.The resulted plasmid was named pFBD-HA-NA-M1.The recombinant transfer plasmid(pFBD-HA-NA-M1)was then transformed into E.coli DH10 Bac competent cell.By transposition,the insert fragment can be transferred to the recombinant baculovirus DNA.Through the blue/white colony selection and PCR identification,the rBacmid-HA-NA-M1 was obtained,which was then transfected into Sf21 insect cells and cultivated at 27?for 72 h to harvest the supernatant.The supernatant were blindly passenged to the third generation.The protein expression and the assmbly of VLPs were identified by western-blot and TEM.The result of TEM showed spherical virus-like particles of about 100 nm in diameter.Western-blot result showed that the protein bands were detected at 62 KD,54KD and 30 KD.In conclusion,virus like particles of H9N2 AIVs composed of three kinds of viral proteins were successfully produced in this study using insect baculovirus expression system,which laid the foundation for the H9N2-VLPs vaccine research in the future.