The Construction Of MDCK Cell Line That Express Siat7e And St3gall Genes Stably

MDCK cells are recognized as one of the most suitable cell lines for the production of AI vaccine. The adherentment of MDCK is too strong to make it suspended, which limits its application in industrial mass production. The siat7e gene was identified as one of the genes that play a role in controlling the degree of cell adhesion. The product of st3gall gene can improve the sensitivity of host cells to avian influenza virus. In this study the siat7e and st3gall genes were transfected into MDCK to obtain a MDCK cell line which can adapt to suspension culture and can produce high titer of avian influenza virus.The siat7e gene and st3gall gene were cloned from human blood sample and chicken blood sample by polymerase chain reaction(PCR), and then were used to construct the pReceiver-siat7e-st3galI express vector. The results of restrictive enzyme digestion、PCR and DNA sequencing analysis suggested that the recombinant expression vector was constructed successfully. The MDCK cells were electroporated with double-gene express vector under150V,5ms condition. The results of observation under fluorescence microscope48h after transfection showed green fluorescence suggested double genes expressed successfully.32monoclonal cells were selected using twice limiting dilution method. Double-gene expression of10monoclonal cells, B3-1、B3-2、B3-3、C5、D5、S5-1、CV9、CV10、 E2、F2,were identified as positive by genome PCR and RT-PCR identification.A relative quantitative PCR was established to detect siat7e gene and st3gall gene. A MDCK cell line (MDCK-C5) expressed double gene highly and stably was selected from10monoclonal cells based the above method and flow cytometry. The H9N2A strain C5was inoculated to adherent MDCK and MDCK-C5cells.The results showed that the hemagglutination titer of virus in MDCK-C5was higher than in MDCK cells.The MDCK-C5cell line was suspend domesticated by gradually decreasing the serum concentration,the cell density of MDCK-C5after suspended domesticated reached to1.3×106cells/mL, and cell viability maintained above60%. The virus was harvested, after48h H9N2A strain C5inoculated to MDCK-C5suspension cells, the EID50of virus content reached to10-569/0.1mL and hemagglutination titer of virus was up to2-8.The MDCK-C5cell line which can adapt to suspension culture and can produce high titer of avian influenza virus was constructed successfully. The research established foundation for industrial mass production.