Preliminary Study On The Multiepitope Vaccine Of Foot-and-Mouth Disease Derived From CHO Cell

Chinese hamster ovary cell(CHO),as a mammalian cell eukaryotic expression system,can not only recombine the natural structure of the protein,but also maximize the biological and immunological functions of the recombinant protein,and has been widely used in biological preparations in medical production,such as drugs,cytokines and monoclonal antibodies.Due to the high production cost,CHO cells have not been widely used in the production of veterinary vaccine antigens and biological agents,nor in the production of FMDV(Foot-and-mouth disease virus)recombinant antigens.At present,the recombinant antigens of most FMD(Foot-and-mouth disease)recombinant subunit vaccines are produced using prokaryotic expression systems,and their natural structure and biological function cannot be guaranteed.Therefore,it is particularly important to develop genetically engineered cell lines that secrete and express the FMDV antigen with a natural structure at a high level,which is also the development trend of vaccine research in the future.In order to achieve this goal,this study used the FMDV type O epitope as the material,CHO cells as the research object,and CRISPR / Cas9-mediated homologous recombination technology as the technical means.Construction of a large number of genetically engineered cells secreting FMDV antigens can provide genetically engineered CHO cell lines for large-scale production of FMDV recombinant antigens with natural structure and function.First,use information technology to predict and screen signal peptides that increase the secretion and expression of O-FMDV epitopes(OME1)in CHO cells,design signal peptide(OME1)fusion genes,and construct p CI-neo recombinant plasmids containing different signal peptides.This plasmid was used to transfect CHO cells to select a signal peptide that can promote secretion and expression of the target protein.WB results show that there are nine signal peptides that mediate the secretion and expression of OME1 fusion gene in CHO cells.Among them,SCGB1D1 isoform signal peptide-OME1 has the highest amount of recombinant protein,which is suitable for CHO cells to secrete FMDV recombinant antigen.Secondly,the SCGB1D1 isoform signal peptide and the reconnected O-type FMDV epitope(OME2)were used as elements,and HBc Ag capable of self-assembling virus-like particles(VLPs)as the backbone,to design the SCGB1D1 isoform-HBc Ag-OME2 fusion gene.The donor plasmids carrying the SCGB1D1 isoform-HBc Ag-OME2 gene was constructed,and Using CRISPR / Cas9-mediated homologous recombination technology to integrate the fusion gene carrying SCGB1D1 isoform-HBc Ag-OME2 into the HPRT hypoxanthine phosphoribosyl transferase gene and the telomere region of chromosome 8 in CHO cell chromosome Construct genetically engineered CHO cells carrying the SCGB1D1 isoform-HBc Ag-OME2 fusion gene.After screening 300 CHO cell islands by resistance pressure,they were amplified and cultured.Immunofluorescence microscopy observed that the cloned CHO cells had a large amount of green fluorescence and identified them.The genome extracted from the cloned CHO cells was used for ligation PCR.Results The SCGB1D1 isoform-HBc Ag-OME fusion gene was detected,indicating that the gene has been successfully integrated into CHO cell,and WB results show that the CHO cell culture fluid contains protein components that can immunoreact with anti-his-tag antibodies and positive sera of FMDV type O,once again proved that the fusion gene was successfully integrated and expressed constitutively.This fully demonstrates that this study successfully constructed a genetically engineered CHO cell line capable of expressing FMD recombinant antigen constitutively.In conclusion,this study not only screened the signal peptide that can promote the efficient secretion of recombinant proteins of FMDV antigen epitopes in CHO cells,but also successfully constructed a genetically engineered CHO cell line that can express FMD recombinant antigen constitutively.This will provide a material basis and new clues for the large-scale production of recombinant antigens of FMD vaccine with natural structure and biological and immunological functions using this cell line.