Establishment Of A Stable Cell Line Secretory Expression Of Zika Virus Non-structural Protein 1 And Exploration Of Cell Immortality

BackgroundZika virus(ZIKV)infection has become a major public health problem in the tropical and sub-tropical regions.Currently,there is no specific anti-Zika medicine,and preventive vaccine is still under researching.Early diagnosis has important significance in preventing ZIKV transmission,and also save valuable time for patients to receive symptomatic treatment.During acute infection phase,virus encoded NS1 protein and release into blood.NS1 protein is highly conserved in different flaviviruses,resulting in been used for differential diagnosis.Among most flaviviruses diagnosis tools,NS1 protein and antibody detection has been the primary choice.Immortalized cell PER.C6 from Crucell corporation was obtained by transfecting human type five adenovirus E1 region coding genes into 18 week-old human embryo retinal(HER)cells.Because of the ability to express foreign genes efficiently and stably,the cell line is widely used in the production of vaccines,recombinant proteins,monoclonal antibodies and gene therapy.However,expensive transfer fees have also deterred most biopharmaceutical companies.ObjectivesThe purpose of this study was to establish the mammalian cell expression system of ZIKV NS1 protein and obtain high-quality NS1 antigen,and lay the foundation for ZIKV serological detection.In order to obtain more advantageous mammalian cell expression tool,this study also preliminarily explored the method for establishing immortalized cell lines.MethodsThe ZIKV NS1 expression gene was cloned into lentiviral vector,and the recombinant plasmid and helper plasmids were transfected into HEK 293T cells correctly.After 48 hours,lentivirus(LV-CMV-EGFP-Zika-NS1)was collected.Then recombinant lentivirus was added to HEK 293T cells,and recombinant monoclonal cells were selected finally.Western Blot was used to detect the secretion of NS1 protein by successive passages of recombinant cell supernatantand.The cell fluoresce-nce rate of the recombinant cell line continuously passaged was detected by flow cytometry.The recombinant protein was purified by affinity chromatography,and the purification effect was analyzed by SDS-PAGE.The purified NS1 protein was immu-nized with BALB/c mice and the serum antibody level was measured by indirect ELISA.Adenovirus El gene expression vector pUC18-PGK-E1-polyA was constructed and transfected into L02 cells.After 48 hours,the transfected cells were diluted 100 times and cultured for 20 days.Selected cell foci with morphological differences from the original cells were identified by Western Blot.ResultsEnzyme digestion and sequencing showed that recombinant expression vector pLV-CMV-EGFP-Zika-NS1 was constructed successfully.After transfection of HEK 293T cells with lentivirus,8 recombinant HEK 293T cell strains expressing recombinant ZIKV NS1 protein were obtained by limited dilution method.The result of Western Blot showed that the highest expression level was found in clone seventh and named HEK-293T-Zika-NS1.The results of Western Blot and flow cytometry showed that the NS1 protein expression level,fluorescence rate and fluorescence intensity of the recombinant cell strain were not significantly changed until thirty-fifth generations.The purity of purified recombinant NS1 protein was over 90%.After the purified recombinant NS1 protein was immunized against BALB/c mice,a high level of anti NS1 antibody was produced.The result of Western Blot showed that the LO2 cells transfected with pUC18-PGK-E1-polyA plasmid expressed adenovirus E1 protein.The cell foci differentiated from the original cells could be observed in the transfected L02 cells.At present,the cell focus that expresses Ad5 E1 protein has not been screened by Western Blot.ConclusionsIn this study,we successfully constructed a recombinant HEK 293T cell strain expressing recombinant ZIKV NS1 protein and obtained recombinant NS1 protein with high purity.The antigen has good immunogenicity and can stimulate the body of mice to produce high-level antibodies.This study laid the foundation for ZIKV diagnostic methods.In addition,the established recombinant cell strain provided a cell model for the biological characterization study of ZIKV NS1 protein.The preparation of immortalized cell by plasmid and lentiviral transfection methods has not been successful yet.It is necessary to improve the existing methods or explore new methods for immortalization of cells.