Construction And Identification Of The Cell Lines Stably Expressing B-type Influenza Virus Hemagglutinin Protein

Objective To construct a stable cell line expressing hemagglutinin(HA)protein of B-type influenza virus.Methods The influenza B virus of Victoria lineage was cultured in MDCK cells,then extracting the RNA and using it as template,the gene of hemagglutinin protein of the virus was amplified by RT-PCR and PCR.After digested by restriction enzyme,the HA gene fragment was cloned into pc DNA3.1(+)eukaryotic expression vector,and then obtained the recombinant plasmid.The plasmid was identified by gene sequencing,PCR assay and restriction enzyme digestion.After that,the plasmid was co-transfected into HEK293 cells with PLV-EF1a-EGFP(2A)Puro plasmid via(Liposome mediated method).The transfected cells were screened with a medium containing puromycin of 0.5?g/m L concentration for three weeks.The monoclonal cell lines were obtained by limiting dilution method.After expanding culture,the expression of HA protein in the cell lines was analyzed by indirect immunofluorescence?Western Blot and flow cytometry.The positive recombinant cells were subcultured for 15 passages with low concentration puromycin.The method of IFA and genomic PCR were used to identify the expression of HA protein and the genetic stability.The cell lines were incubated with human,which contained influenza B virus neutralizing antibody,and then analyzed by flow cytometry.Results Firstly,HA gene was obtained by RT-PCR and PCR assay,and the sequence of it was verified correct.Secondly,the recombinant plasmid pc DNA3.1(+)-HA was identified correct and HA gene fragment was confirmed successfully cloned into the plasmid.Thirdly,The positive cell clones were screened out by puromycin after transfected into HEK293 cell line.The specificity of the recombinant protein was tested by IFA and western blot assays.The expression of HA protein and the stable inheritance of the gene were detected by indirect immunofluorescence and PCR reaction after successive 15 generations of subculture.The results from the flow cytometry has a statistically significance.Conclusion The cell lines expressing the hemagglutinin protein(HA)of influenza B virus were Successfully constructed,the cell line can be stably expressed HA antigen with immunological activity of influenza B virus on the HEK293 cell surface.It would be helpful to-lay a good foundation for further HA protein functional research.