Study On The Technology Of Serum-free Full Suspension MDCK Cells Culture Of H9 Subtype Avian Influenza Virus

Serum-free full suspension MDCK cells culture of H9 subtype avian influenza virus is a culture method aiming to overcome the shortcoming of chicken embryo and adherent cell used in avian influenza vaccine production,however,the current process of serum-free suspension MDCK cell culture of H9 subtype avian influenza virus is not yet mature,therefore,this paper explores the process of cultivating H9 subtype avian influenza virus to provide experimental data support and reference for the establishment and optimization of cell bioreactor technology for large-scale production of avian influenza vaccine.In this study,we first investigated the growth characteristics of MDCK-sus cells in serum-free full suspension culture in shaking flasks,the influence of infection parameters on virus proliferation,changes of nutrient metabolism of AIV in the proliferation of MDCK-sus cells,and effects of different proportions of fresh culture medium on viable cell density and virus proliferation at different time.The results showed that 1.5*106 cells/m L inoculation density had the best efficiency;TPCK-trypsin with the final concentration of 7?g/m L,culture temperature of 33?,MOI of 0.01 and TOI of 60 hours were suitable,the HA titer of avian influenza virus can reach to 10 log₂,and the virus content can reach to10^8.21±0.61EID₅₀/0.1 m L after optimization;avian influenza virus accelerated the consumption of nutrients such as glucose and glutamine in the process of cell proliferation,and also accelerated the production of metabolic products such as lactic acid and ammonium ions;After adding fresh culture medium and inoculating virus in each proportion group,According to the 3:2 cell suspension and fresh medium volume ratio of adding fresh medium was optimal.The bioreactor process with different specifications was further tried,through the amplification culture to 3 L stirred bioreactor to verify and explore the effect of p H on the proliferation of AIV,continue to enlarge to 50 L torrentiol bioreactor to verify.The results showed that when p H value was 7.0,the titers of HA reached a peak value of 11 log₂and the virus content reached a peak value of 10^9.14±0.10EID₅₀/0.1 m L at 48 hours after infection;suspension cell culture and virus culture technology based on 3 L stirred bioreactor continued to scale up to 50 L bioreactor:the HA titers reached the peak value of 11 log₂and the virus content reached the highest value of 10^8.76±0.10EID₅₀/0.1 m L 48 hours after infection,which was basically consistent with the culture characteristics of 3 L bioreactor.In order to increase the surface receptor abundance of MDCK cells and further improve the titer of avian influenza virus,in this study,chicken ST3GAL1 gene was inserted into lentivirus vector to construct recombinant lentivirus,then transduced cells,after purinomycin screening,resistant cells were collected and named as MDCK-adh-st3gal1 cells;MDCK-adh-st3gal1 cells were passed on to the tenth generation,the expression of ST3GAL1 gene in chicken MDCK-adh-st3gal1 cells was detected by q PCR,and the abundance of SAa-2,3Gal and SAa-2,6Gal receptors on the surface of MDCK-adh-st3gal1 cells was detected by flow method,finally,the cell proliferation ability of H9 subtype avian influenza virus before and after modification was compared.The results showed that the recombinant plasmid transfected the cells well with a fluorescence rate of over 80%;MDCK-adh-st3gal1 cells were passed on to the tenth generation,and the target gene chicken ST3GAL1 was still expressed;MDCK cells stably expressing chicken ST3GALI gene which increased the abundances of a-2,3-linked receptors on cell surface and were more suitable for the infection of H9 subtype avian influenza virus.The production technology of H9 subtype avian influenza virus in serum-free full suspension of MDCK cells in bioreactor and the modification of MDCK cell line were studied,which provide a reference for the establishment and optimization of cell bioreactor for large-scale production of avian influenza vaccine technology.