Study On The Expression Of Tree Shrew Sialyltransferase And The Interaction Between Recombinant Cells And Influenza Virus

Influenza virus is an infectious agent that can cause severe respiratory infections in humans and animals and continues to pose a threat to public health.To understand the pathogenic mechanism of influenza viruses,the use of in vitro cell models and small mammalian models has become indispensable.However,the cell lines and animal models currently used in laboratory environments have many flawed and inadequate.Sialyltransferase(ST)regulates the generation of sialic acid receptors combined with viral hemagglutinin is the key to the effective infection and transmission of influenza virus,but sialic acid receptors and ST substrates exist in common cell lines and animal models of influenza.The difference between the substance(CMP-SA)and humans is large,and it cannot accurately reflect the characteristics of human influenza diseases.In this study,9 commonly used cell lines(MDCK,BHK,HEP-2,16HBE,A549,Vero,CHO-k1,DF-1 and 293T)were selected,and the human/avian influenza viruses were compared in different cases by half the tissue infection dose(TCID50).Growth and replication levels in cell lines;direct immunofluorescence method to detect the distribution of sialic acid receptors on the cell surface and its binding to influenza virus;fluorescence quantitative RT-PCR method to detect the m RNA transcription level of sialyltransferase(ST3GAL?;ST6GAL?).The results showed that all 9 cell lines contained sialic acid,among which MDCK and DF-1 cells had the highest expression level of sialyltransferase,the most abundant surface sialic acid,the largest amount of bound virus and the highest sensitivity to influenza virus;the sialic acid content of BHK cells.The lowest amount of influenza virus that can be combined,and low sensitivity to influenza virus.Among the 9 commonly used cell lines in the laboratory,the sialyltransferase gene expression level and the sialic acid content on the cell surface were positively correlated with the amount of influenza virus bound.In the previous research,it was found that tree shrews can effectively infect influenza viruses and have good symptom simulation.The ST gene sequence and sialic acid receptor are closer to humans than the original model animals.On this basis,this subject detected ST expression in tree shrew respiratory tract tissues,selected ST(ST3GAL?/?;ST6GAL?)related to influenza virus and constructed their expression plasmids to transfect BHK cells,and screened out the cell line for strew ST gene expression.Reel-time PCR method was used to detect the sialyltransferase(ST3GAL?/?;ST6GAL?)m RNA transcription level and Western blot method to detect the target protein expression,which proved that tree shrew ST was expressed in BHK cells at the gene and protein levels.Direct immunofluorescence method was used to detect the sialic acid receptor on the cell surface.The recombinant cell BHK-tu STs cell surface sialic acid flu receptor was significantly increased.TCID₅₀ compared the growth and replication levels of human/avian influenza virus in BHK-tu STs at 12 h,24 h,48 h,and 72 h,and the plaque test of human/avian influenza virus on BHK-tu STs cells.It proves that BHK-tu STs recombinant cells are more susceptible to influenza virus tropism and infection replication than the original BHK cell line.Based on the above results,it is verified from the cellular level that the tree shrew ST can effectively mediate the adhesion and replication of influenza virus,which provides an important scientific basis for further clarification of the tree shrew as an animal model of influenza.