Construction And Functional Study Of MDCK Cells Stably Expressing Siat7e Gene

The traditional influenza vaccines are produced by chicken embryo,which has long production circle,cumbersome operation and highly mutation rate of egg-grown viruses.Cell-based influenza vaccines are more similar in antigenicity with original virus,so that the mammalian cells are promise to replace the chicken embryo as a new influenza vaccine production substrate.Clinical trials results showed that MDCK cell culture-derived vaccines were superior to egg-based vaccines in safety and tolerability.However,MDCK cell is anchorage-dependent,which is not conductive to large-scale production.Besides,the serum required for cell culture has disadvantages of batch instable and composition unclear.It's the general trend to produce biological products using suspension cells that can proliferate in serum-free medium.Studies have showed that the gene siat7e could effectively attenuate cell adherence.In this study,genetic engineering was adopted to construct the monoclonal MDCK cell stably expressing the gene siat7e,and acclimated into single MDCK-5G7-S suspension cell.After H3N2vaccine strain inoculated,the yield of virus in suspension cells is significantly higher than that of adherent cells,laying the foundation for the MDCK suspension cells to produce influenza vaccines in large scale.In the first part,stable cell line of MDCK expressing gene siat7e were obtained.The expression vector pcDNA3.1-siat7e/Zeocin was constructed firstly and transformed into MDCK cells by electroporation.After screening with Zeocin and limiting dilution method,monoclonal MDCK cell expressing gene sait7e were obtained.Then,the expression of gene siat7e was identified from mRNA level and protein level by RT-PCR and WB methods,and the relative expression level of gene siat7e was analyzed by qRT-PCR method to obtain stable cell line MDCK-5G7 with high expression of gene siat7e.In the second part,MDCK-5G7 cells were subjected to suspension acclimation and functional studies.First,the four serum-free mediums,OptiPRO SFM,FreeStyle293,CD293 and VirusPro MDCK-S,were screened,and the VirusPro MDCK-S medium which can rapidly adapt MDCK-5G7 cells was selected to acclimate MDCK-5G7 cells,obtaining MDCK-5G7-S suspension cells growing in VirusPro MDCK-S.The initial inoculation density of the cells was optimized to determine the optimal seeding density of 1×10⁶ cells/mL.And the highest cell density of 2.45×10⁶ cells/mL was obtained at 96 h while the survival rate was above 80%always.The vaccine strain A/Switzerland/9715293/2013?H3N2?was inoculated with adherent MDCK,adherent MDCK-5G7,MDCK-S suspenion and MDCK-5G7-S suspended cells at multiplicity of infection of 0.1.Collecting samples at 12h,24h,48h,72h and 96h,Measuring the CCID₅₀ titer,and drawing the virus growth curve.It was found that the virus had the similar proliferation trend in the four types cells.Comparing the virus titer of 48h,the virus yield of suspension cells was higher than that of adherent cells,and the transfer of siat7e gene further increased the titer.The virus titer CCID₅₀0 of MDCK-5G7-S could reach 10^7.9/mL,which provides new ideas for influenza virus research.