Objective To construct a new cell line by overexpressing fucosyltransferases 2(FUT2)gene in human embryo kidney-293T(HEK-293T)cells,FUT2 has the founction of catalyzing the formation of Histo-blood group antigen(HB-GA),which is one of receptors of norovirus.The constructed cell line was used to study interactions of norovirus and its re-ceptor.Methods FUT2 lentivirus plasmid was constructed,and the FUT2 lentivirus plasmid,pMD.2G and psPAX2 packa-ging plasmids were co-transfected into HEK-293T cells with Lipofectamine 2000.Lentivirus particles were harvested and in-fected HEK-293T cells at logarithmic growth stage,to obtain HEK-293T/FUT2 cell lines by purinomycin pressure screening.PCR was used to identify whether the FUT2 gene was integrated into the genome.The mRNA transcription level of FUT2 in HEK-293T/FUT2 cells was detected by RT-qPCR,and the expression of HBGAs in HEK-293T/FUT2 cells was analyzed by flow cytometry.Further more,the binding activity of characterized HEK-293T/FUT2 cells with norovirus virus-like particles(VLP)was analyzed by indirect immunofluorescence.Results The lentivirus plasmid PLVX-IRES-Puro-FUT2 with cor-rect sequence was successfully constructed and lentivirus particles were obtained.HEK-293T/FUT2 cell lines were obtained after lentivirus particles were infected into HEK-293T cells.PCR verification showed that FUT2 gene was successfully integrated into the genome of HEK-293T cells.The level of FUT2 mRNA in HEK-293T/FUT2 cells was increased by 330 times,and 99.9%of HEK-293T/FUT2 cells expressed H2 Histo-blood group antigen.Finally,GI.1 type recombinant norovirus VLP were able to bind to HEK-293T/FUT2 cells,and EC50 was 2.007 μg/mL.Conclusion In this study,HEK-293T/FUT2 cell line with stable and high expression of H2 His-to-blood group antigen was constructed for the first time,and a preliminary evaluation method for the detection of GI.1 re-combinant norovirus VLP binding activity was established based on this cell line.
Histo-blood group antigen(HBGA)Human embryo kidney-293T cell(HEK-293T cell)Norovirusesα-1,2 FucosyltransferasesBinding activity
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