Constuction Of BHK-21Cell Line Stably Expressing Chicken ST3GALⅠGene And The Proliferation Of Avian Influenza Virus

Bird flu (AI) is a disease caused by influenza A virus in poultry. If chickens,turkeys, ducks and other poultry waterfowl, wild birds infection, it will cause poultrydeaths, egg production decline, serious respiratory disease syndrome or recessiveinfection, system and many kinds of disease. AI virus spreaded through the surfacehemagglutinin (HA) infecting host cell binding cell surface sialic acidoligosaccharides receptor. Avian influenza viruses preferentially bind tosialyloligosaccharides containing terminal N-acetyl sialic acid linked to galactose byan α-2,3-linkage (NeuAc α-2,3-Gal), while human influenza viruses mainly bind tothose containing an α-2,6-linkage (NeuAc α-2,6-Gal).The chicken ST3Gal Ⅰ gene was coloned and incorporated into the eukaryoticexpression vector pcDNA3.1-EGFP to generate the recombinant plasmid. Therecombinant vector pcDNA3.1-EGFP-ST3Gal Ⅰ was transfected into BHK-21cellsusing lipofectamine2000. We got monoclone cell lines by G418resistance screeningand RT-PCR appraisal. Green fluorescence could be observed. BHK-21-ST3Gal Ⅰ cellexpressed higher amounts of2,3-linked receptors than parent BHK-21cell by Flowcytometryt analysis. Continuous passing five generations and ten generations can stilldetected the expression, so ST3Gal Ⅰ gene was stably expressed in cells. To screenproliferation of avian influenza virus(AIV) H9Subtype and H5N1Re-5in BHK-21-ST3Gal Ⅰ cell, We according to the different proportion of the H9subtype andH5N1Re-5avian influenza virus vaccinationed BHK-21-ST3Gal Ⅰ cells and BHK-21cells, at the same time we had ordinary pancreatic enzyme group and TPCKpancreatic enzyme control group. Harvested cell liquid after72hours, we usedconventional method for determining hemagglutinin (HA) titer. The experimentalresults showed that the HA titer of influenza virus of BHK-21-ST3Gal Ⅰ cells reached1:256, significantly higher than BHK-21cell group, and showed that the moresuitable for the flu virus proliferation, having the potential of the influenza virusvaccine production.Screening to stable expression of genes ST3Gal Ⅰ BHK-21celllines, but for the further study of the bird flu virus receptors characteristics andlarge-scale production of vaccine cell culture lays the foundation.