Influenza A H1N1 And A H3N2 Influenza Virus In MDCK Cell Line Study On The Characteristics Of Proliferation

At present,large-scale animal cell bioreactor culture technology is widely used in the production of viral vaccines.The influenza virus cell vaccine produced by this technology abroad has passed the safety certification and has been widely used in clinic.The production of influenza vaccine in China is still mainly based on backward chicken embryo technology.Chicken embryo seedling has been gradually eliminated in production practice because of its high labor intensity,low efficiency,small batch,easy pollution,high cost and the shortage of SPF embryo supply and poor emergency response ability.In order to improve the production,quality and market competitiveness of influenza vaccine,it has become an urgent technical difficulty for vaccine manufacturers in China to develop the technology of large-scale cultivation of animal cells to produce influenza virus cell vaccine in bioreactor as soon as possible.At present,in the development of influenza cell vaccines,vaccine companies have found that in large-scale cultured animal cells,influenza virus proliferation efficiency is low,and can not reach the required viral titer for vaccine preparation in a short time,which limits the development speed of influenza virus cell vaccines.It is reported that canine kidney-derived MDCK cells are sensitive to influenza virus and have been used in veterinary vaccine production.In this paper,two strains of influenza A H1N1 and H3N2 vaccine viruses were multiplied and amplified in SPF chicken embryos,respectively,to construct the working strain library,and to carry out biological identification.Then culture,proliferation adaptation and passage domestication were carried out on MDCK adherent cells and suspension cells.The conditions and rules of culture and proliferation of two influenza viruses on MDCK cell lines were explored.The optimum multiple viral infection?MOI?,the optimum time of inoculation and receipt were determined,and the haemagglutination titer?HA?and disease of the two influenza viruses after proliferation on MDCK adherent cells and suspension cells were increased.Drug titer(CCID₅₀).When H1N1 strain proliferates in MDCK adherent cells and suspension cells,HA can reach 2⁸HA units/25?l;CCID₅₀ can reach7.50TU/ml and 7.32 TU/ml;When H3N2 replicates in two kinds of MDCK cells,the HA of the obtained venom is slightly lower than that of H1N1 cells,the highest is 2⁷HA units/25?l,and the highest is CCID₅₀ can reach 7.12 TU/ml and 6.79TU/ml.Respectively,which can satisfy the antigen content of vaccine production requirement.Finally,according to the optimized parameters of virus culture conditions,the two strains of virus were cultured and amplified on MDCK adherent cells and suspension cells,then collected and frozen for biological identification.The virus substitution bank and seed virus bank of the two viruses were established,which laid the foundation of virus for the subsequent production of influenza virus cell vaccine by using MDCK cells cultured in bioreactor,and there are great significance for the development and production of influenza virus cell vaccine based on MDCK cells.