| Avian influenza is one of the main diseases in poultry.Every year,there are several sporadic incidents all over the world,which have a serious impact on the poultry industry.It not only causes huge economic losses to human beings,but also causes public security problems.Vaccination is an effective way to prevent and reduce the occurrence of avian influenza.The traditional avian influenza vaccine is mainly chicken embryo vaccine.There are some disadvantages in the production process of chicken embryo vaccine,such as large amount of eggs,low yield,long production cycle,complicated process and difficult to realize industrial production.Compared with chicken embryo production process,cell production process has the advantages of simple process and continuous operation,especially the rapid development of cell suspension culture technology,which makes the advantages of cell matrix production vaccine more obvious.Suspension culture technology has the advantages of easy control of culture conditions,easy monitoring of cell growth and high level of industrialization.It can achieve efficient replication and large-scale production of avian influenza virus,and has important application value.In this study,adherent MDCK cells were used as the research object.On the basis of acclimation to serum-free culture,adherent MDCK cells were cultured in shake flask shaker system,and then cultured in bioreactor.The low pathogenic H9subtype avian influenza virus was inoculated into suspension MDCK cells for adaptive culture.The antigen cultured in cell matrix was prepared into inactivated vaccine to immunize animals,and the immune effect of cell-derived vaccine was evaluated by animal experiments.The adherent MDCK cells were acclimated in serum-free medium with serum concentration of 10%,5%,3%and 1%,respectively.Finally,the adherent cells were cultured in serum-free medium for 30 generations.The cell density was 1.2×10~6cells/ml,the viability was 95%,and the doubling time was 28 h.There was no significant difference between the medium with 10%serum content and the cell density of 1.0×10~6 cells/ml.Suspension culture of adherent MDCK cells was acclimated in shake flask shaker system.After 40 generations of suspension acclimation,the cell density was 2.0×10~6 cells/ml and the activity was more than 90%.It was named MDCK-XF.Eight monoclonal cell lines were screened out from 2 rounds of cell clones by limited dilution method.The cell density was 3.2×106 cells/ml,the viability was more than 92%,and the doubling time was 20 h.Compared with the suspension cells initially screened,the cell density and viability were significantly improved.The cell line was named mdck-xfl.According to the requirements of cell bank,the MDCK suspension cell primitive cell bank was established.The optimal culture conditions of suspension mdck-xfl cells were determined as follows:inoculation density(0.5～0.7)×106 cells/ml,p H 7.2～7.6.The results showed that cell density and viability were significantly increased by using serum-free medium B and optimized culture conditions.After 72 hours of culture,the cell density reached 4.0×106 cells/ml and the survival rate was more than 94%.The cell density was 4.0×106 cells/ml and the viability was more than 94%after 72 h in a 7.5 L bioreactor.It was the same as that of shaking flask culture and could be stably passaged.The selected cells were suitable for large-scale culture.The low pathogenic H9 subtype avian influenza virus was inoculated into adherent and suspension MDCK cells respectively,and the pathological changes were obvious.When MOI was 0.01,virus collection time was 72 h,and TPCK trypsin concentration was 4μg/ml,the highest viral HA was 9 log2.Compared with that before optimization,the viral ha titer increased by 1-2.The HA titer of HA was 1-2higher than that of adherent cells and chicken embryo culture.The results of animal experiment showed that the geometric mean value of hi produced by immunized cell-derived vaccine group was 6 log2 on 21 days.After challenge,throat swabs and cloacal swabs were collected.The virus was not isolated from chicken embryos after inoculation,and the protection rate was 100%.It was equivalent to that of chicken embryo vaccine and had good immune effect.In this study,high density suspension cell lines were obtained by suspension acclimation and monoclonal screening in serum-free medium,which was suitable for scale-up culture in bioreactor.This cell line can efficiently propagate low pathogenic H9 subtype avian influenza virus.The prepared inactivated vaccine can induce the body to produce high antibody and has good immune protection.It has achieved the same level of immune effect as chicken embryo vaccine.It lays the foundation for the large-scale production of avian influenza vaccine by MDCK cells,and provides theoretical basis and research for the large-scale production of other virus vaccines thinking. |
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