Study On Assembly Of H7N9 Subtype Avian Influenza Virus-like Particles By Recombinant Multibac System

In 2013,a new H7N9 avian influenza virus outbreak occurred in East China.As of2017,a total of 1398 cases of H7N9 avian influenza were reported in China,and there were about 500 deaths.The cases covered more than 20 provinces and cities in China.Despite a series of disease prevention and control measures taken by the state,cases of human cases caused by the H7N9 avian influenza virus are still scattered.Faced with such a severe situation,the development of a safe and efficient new bird flu vaccine has become the key to the prevention and control of H7N9 subtype avian influenza.As an ideal candidate vaccine,virus-like particles(VLPs)have the advantages of good immunogenicity,no infection,high stability,and low inactivation,which makes them highly research in the development of viral vaccines.value.A large number of research reports on virus-like particles have been reported at home and abroad.The human papillomavirus-like particles vaccine has been marketed in Europe and the United States,showing a good application prospect of virus-like particle vaccine.The avian influenza virus-like particle vaccine contains no viral nucleic acid,has no biosafety risk when used,and can induce cellular immunity and provide immunoprotection to the same type of heterologous virus.It is currently the most promising alternative to the existing avian influenza vaccine.Therefore,the development of avian influenza virus-like particle vaccine has extremely important scientific and social benefits.This study firstly conducted a wide-scale epidemiological investigation on the prevalence of H7N9 subtype avian influenza virus in China,and then screened out the epidemic strains as vaccine strains,and carried out the main structural proteins HA,NA and M1.Codon optimization and whole gene synthesis as antigen genes,then the HA gene and M1 gene are ligated into the pYBDM-IM transfer vector by conventional digestion and cloning,and the NA gene is ligated into the pUCDM-XIGP transfer vector by conventional enzymatic cloning.The obtained recombinant transfer vector was identified by enzyme digestion,and the specific target band was found by agarose gel electrophoresis,indicating that the recombinant transfer vector has been successfully constructed,and the sequence was further sequenced to further verify whether the antigen gene was correct.The two correct transfer vectors,pYBDM-IM-HA-M1 and pUCDM-XIGP-NA,will be simultaneously recombined into SW106Am/asd~-/InvC~+competent cells by transposition and site-specific recombination,and resistance is taken.The positive recombinant bacterial liquid was screened by screening,blue-white screening and bacterial liquid PCR,and the positive recombinant bacterial liquid was named Am-HA-NA-M1-IM-XIGP.The recombinant bacterial solution was directly infected with Sf9 insect cells by the bacterial infection method established in our laboratory,and the recombinant virus BV-HA-NA-M1-IM-XIGP was harvested.Because the recombinant virus carries both GFP and mCherry reporter genes,it is fluorescent.Red fluorescence and green fluorescence can be observed simultaneously under the microscope.Viral genomic DNA was collected from the supernatant of virus-infected Sf9 cells and detected by specific primers.The results showed that the genomic DNA was PCR-amplified with a specific target band,indicating that the HA gene,NA gene and M1 gene have been successfully Recombination into the baculovirus genome;in addition,the virus-infected Sf9 cell pellet was collected for SDS-PAGE electrophoresis and Western blot,and the expression of HA protein,NA protein and M1 protein was detected by specific antibodies carrying the tag of each of the three genes of interest.In the case,the protein bands of 63KD,53KD and 29KD were found to indicate that the HA protein,NA protein and M1 protein of H7N9 subtype avian influenza were successfully expressed in Sf9 insect cells.The recombinant virus was heavily infected with Sf9 insect cells,and virus-infected cells were collected for protein purification 5 days later,and then the purified protein was observed under a transmission electron microscope to find a virus-like particles structure having a diameter of 80 to 120 nm.This indicates that the M1,NA and HA proteins are well expressed in Sf9 insect cells and assembled into VLPs.This further demonstrates that the successful use of the baculovirus expression system to produce H7N9 subtype avian influenza virus-like particles provides technical support for the subsequent production and preparation of H7N9 subtype avian influenza vaccine.