Cloning And Expression Of Antibody Gene Of Anti-PEDV Hybridoma Cell Line

Objective: To clone the full-length light and heavy chain gene of antibody from anti-Porcine epidemic diarrhea virus(PEDV)hybridoma cell line and express it in eukaryotic cells.To establish a platform for preparing monoclonal antibodies against PEDV in vitroMethods: Monoclonal antibody subtypes were identified by antibody subtype identification kit.The titer and specificity of anti-PEDV monoclonal antibodies were detected by indirect ELISA.The stability of hybridoma cell lines was evaluated by the analysis of chromosome number and the change of antibody secreting ability by indirect ELISA.The genes of light chain and heavy chain were obtained from hybridoma cell lines by RT-PCR,and expression plasmids PIRES2-Zs Green1-H and PIRES2-Zs Green1-L of the heavy chain gene and light chain gene were constructed by enzyme digestion and ligation.PIRES2-Zs Green1-H and PIRES2-Zs Green1-L were co-transfected into CHO-K1 cells,and the transcription of antibody gene in CHO-K1 cells was identified by RT-PCR,and the expression of antibody gene in CHO-K1 cells was identified by Western Blot.The binding activity of the genetic engineered antibody was detected by indirect ELISA and Western Blot.The human albumin signal peptide,human kappa light chain signal peptide,mouse kappa light chain signal peptide and Gaussia luciferase signal peptide were substituted for light chain parent signal peptide to optimize the signal peptide.Results: The heavy chain subtype of anti-PEDV monoclonal antibody was IgG2 a subtype,and the light chain subtype was Kappa subtype.The antibody titer was about 1:8000,the antibody did not cross-react with PRV,CSFV,PRRSV,TGEV and PDCo V.After 3 months of continuous culture,the number of chromosomes in the hybridioma cells remained constant at 96.The indirect ELISA results indicated that the ability of secreting antibodies of hybridioma cells did not change during the continuous culture.Agarose gel electrophoresis and sequencing results showed that eukaryotic expression plasmids PIRES2-Zs Green1-H and PIRES2-Zs Green1-L were successfully constructed.The results of RT-PCR indicated that the light chain gene and heavy chain gene were all transcripted in CHO-K1 cells,and the results of Western Blot indicated that the antibody genes were successfully expressed in CHO-K1 cells.ELISA and Western Blot results showed that the anti-PEDV genetic engineering antibody had good biological binding activity.The results showed that the ability of mouse Kappa light chain signal peptide to mediate antibody secretion was slightly better than that of the parent signal peptide.Conclusion: Anti-PEDV monoclonal antibody has good specificity and affinity.The antibody gene was successfully cloned and its transient expression was realized in CHO-K1 cells.The results of optimizing the light chain signal peptide showed that the antibody secretion of mouse light chain Kappa signal peptide was slightly better than that of the parent signal peptide.