The Establishment Of R-MDCK Cell Line That Stably Express ST3GAL ? Gene

The receptor of the influenza virus is sialic acid(SA)with the sugar chain on the cell surface.As the composition of conjugated sugar content on the cell surface,sialic acid belongs to the family of the nine carbon sugar molecules.It usually lying in the Most lateral capped position of sugar chain of glycoproteins on the cell surface or glycolipids molecules or other polysaccharide,which carry the negative charge.It plays an important role in the recognition among cells and the interaction between cells and extracellular matrix.Sialic acid is connected to galactose or hexose of the end of the sugar chain with alpha 2,3 or alpha 2,6 glycosidic by its second carbon atoms,among which the Galactose abbreviates Gal.The unique connecting format of sugar and sialic acid is completed by Sialic acid transferase.ST6 Gal transferase mediated sialic acid with alpha 2,6 glycosidic bonds to galactose,while ST3 Gal transferase mediated sialic acid with alpha 2,3 glycosidic bonds to galactose.According to the research,mammalian cells express both SA alpha 2,3 Gal and SA alpha 2,6 Gal.Avian influenza virus attack the host cell through its surface hemagglutinin(HA)bonding with sialic acid oligosaccharides receptors on the host cell surface.Most bird influenza viruses tend to identify the receptor of SA alpha 2,3Gal,while human influenza viruses tend to identify the receptor of SA alpha 2,6 Gal,swine influenza viruses distinguish both of them.However alpha 2,3-galactose receptors of mammals cell membrane are low abundance,which lead to low titer of the growing of the low pathogenic avian influenza virus in the cells and have a serious influence on The industrial process that passage cells replace chicken embryo to produce low pathogenic avian influenza virus vaccine.Foreign scholars have build stable expression cell lines by injecting human influenza receptors component ST6 GAL I into MDCK cells,which research human influenza virus affinity ability to2,6 connectivity receptor.Domestic Mr.Bu have also built the recombinant MDCK cells that is suitable for the growth of the highly pathogenic avian influenza virus,however titer of the low pathogenic avian influenza virus is low.Therefore,screening out the MDCK cells lines that can effectively improve the low pathogenic avian influenza virus titer and stably express chicken ST3 Gal I gene is particularly important.Given this,chickens ST3 GAL transferase gene is optimized in this study,The method is to clone the chicken ST3 Gal I genes and insert it into eukaryotic expression plasmid p IRES,and construct recombinant plasmid p IRES-ST3 GAL I,By PCR amplification,restriction enzyme digestion and DNA sequence analysis purpose gene was identified the correct in the right direction and the inserting size,it was successful in building p IRES-ST3 GAL I eukaryotic expression vector.Eukaryoticexpression vector p IRES and recombinant expression vector transform into e.coli DH5? with different concentration IPTG induction,from which we found that the different concentration IPTG induced purpose protein yield have no significant differences between low tendency 0.2 mol/ L and 1.2mol/ L IPTG also can induce to produce a large number of target protein.SDS-PAGE showed that the target protein have expressed.Eukaryotic expression vector which was identified the correct transfect MDCK cells,and use G418 resistance to screen stable expression recombinant R-MDCK cells.PCR and RT-PCR technique identify monoclonal cell lines which express positive.Different H9 subtype avian influenza virus strains in the same titer vaccinate R-MDCK cells and control group MDCK cells,so different H9 subtype avian influenza virus strains on the R-MDCK cells proliferation effect was tested by HA test and TCID50 test.Results were that HA and TCID50 results of 4 H9 subtype avian influenza virus strains in R-MDCK cells were obviously higher than that of control group MDCK cells.ST3GAL?gene in the 12 th generation R-MDCK cells can still stable expression,and R-MDCK cells can improve effectively culture titer of H9 subtype low pathogenic avian influenza virus,and then can be used instead of chicken embryos to produce H9 subtype low pathogenic avian influenza virus.