Development Of H5N1 Avian Influenza Vaccine Based On MDCK Suspension Cells Culture

The outbreak of influenza has brought significant losses to the development of social economy and poses a serious threat to people's lives.At present,inoculation of influenza vaccine is the most effective means to prevent influenza.Current influenza vaccines are predominantly produced by egg-based production methods,the existence of its own defects led to the inability to manage the possible threat of massive outbreaks of influenza.With the development of animal cell culture technology,the production technology of influenza vaccine based on animal cell culture has gradually become the best choice for replacing egg-based production methods because of the advantages of safety and high production capacity.However,the virus originated in chick embryo may not be able to produce enough energy to infect the animal cells in the process of upgrading egg-based influenza vaccines into cell culture-based influenza.On the other hand,there are more operational and control parameters need to be optimized in the process of producing cell culture-based influenza compared with the egg-based production.In this paper,the object of study are the H5N1 avian influenza virus,culture environment and the conditions of infection.First high-quality virus is obtained by domesticating virus originated in chick embryo.The results showed that Tissue culture infective dose of virus increased to(7.50±0.25)1g TCID50/0.1 ml after domestication in suspension cell system under Multiplicity of infection equaled 0.0001 and TPCK-trypsin concentration of 5 mg/L.The effects of pH on Hemagglutinin titer,as well as the Cell density at infection and TPCK-trypsin on the growth of cells and viral yield were investigated.The results showed that the virus yield was increased by 68.2%when the pH was adjusted to(7.2±0.2)by adding the standard NaOH solution with concentration of 1.33 ml/L every 12 h after infection.When Cell density at infection is 7.0×106 cells/ml,The Hemagglutinin titer of virus increased to 211.5±0.25 HA units/25 ?l under Multiplicity of infection equaled 0.0001,TPCK-trypsin concentration of 14 mg/L.Finally,the technological parameters were tested on the 7 L bioreactor,and the Cell-specific virus yield reached(2.12±0.44)×104 virions/cell.The level of hemagglutination inhibition antibody reached 26.80±0.50 HI units/25 ?l after 21 days of immunization.Through the research of this paper,the production of influenza vaccine based on MDCK suspension cell culture was developed,which also provides beneficial reference to animal cell culture-based influenza vaccine development.