Establishment And Preliminary Application Of Methods For Purification H9N2 Subtype Avian Influenza Virus And Quantification Of Its HA Protein

H9N2 subtype avian influenza is a zoonotic infectious disease caused by H9N2 subtype avian influenza virus.It is widely spread around the world and seriously endangers the development of poultry industry and human health.Vaccination is the most effective way to prevent and control avian influenza.HA protein is the main antigen on the surface of avian influenza virus that can induce immune response.At present,in the upstream and downstream processes of H9N2 subtype of avian influenza virus vaccine,HA experiment is the only method used in characterization of total production of the virus.However,this method has some defects,such as low accuracy and failure to absolutely quantify.Meanwhile,the early stage of the laboratory data showed that HA titer could not accurately reflect the immune efficacy of vaccine.Therefore,it is necessary to establish a quantitative method for the HA protein of H9N2 subtype avian influenza virus,so as to provide relevant quality attribute indicators for the development and production of vaccine.The virus was firstly isolated and purified.After clarified by differential centrifugation,virus particles in the supernatant could be completely precipitated under the optimized conditions:PEG6000 at a final concentration of 8%(W/V)and centrifugal force of 6000 g.The viruses were mainly concentrated at 40%-45%after separation by 20%-60%(W/V)continuous sucrose gradient.The virus particles collected were intact and relatively pure.An improved automatic collection system was used to collect virus centrifugal zone.The final HA recovery of optimized purification processes of virus was up to 79.6%.Secondly,the virus was deglycosylated by using PNGase F.The result showed that a viral protein concentration of 1000 ?g/ml and PNGase F enzyme/substrate ratio of 1/40(v/v)were ideal for the virus sample as demonstrated by clear and distinct viral protein bands on the gel.The HA ratio could be determined by gray analysis of four major viral protein bands(NP?HA1?M1 and HA2)after deglycosylation by using Image J software.Three batches of inactivated H7N9 virus vaccines were used for accuracy verification of this gray analysis method.The results showed that the relative deviation between the determination result of this method and SRID value was no more than 10%.Finally,the purification and quantitative methods established above were used to analyze the HA contents of H9N2 subtype avian influenza viruses produced by different culture systems and processes.The results showed that under the same HA titer,the HA contents of viruses produced by different systems were different.Meanwhile,harvest time,MOI,maintenance medium and cell passage influenced the HA content of viruses.In this paper,Simple methods for purification of H9N2 subtype avian influenza virus and quantification of viral HA protein were successfully established,providing relevant quality attribute indicators for the development and production of H9N2 subtype avian influenza virus vaccine,in guarantee under the premise of "high quality" the pursuit of high yield.