Purification And Immunogenicity Study Of Recombinant SARS-CoV-2 S Protein Trime

Objective: To construct a Trimer Domain Antibody(TD-Ab)for purifying the Prefusion-Delta(PF-D)protein and to study the immunogenicity of the PF-D protein.Methods: The pEE12.4 TD-Ab recombinant plasmid was constructed by homologous recombination.The pEE12.4 TD-Ab recombinant plasmid was transfected into CHOK1 cells through electroporation,and stable transfected cell pools expressing TD-Ab were constructed by screening with methionine sulfoxide(MSX)using the glutamine synthetase(GS)system;The purified TD-Ab was coupled with NHS-activated Sepharose to construct a special filler for purification of PF-D protein;Study the immunogenicity of PF-D protein by immunizing C57BL/6 mice and using ELISA and ELISpot for detection;C57BL/6 mice were immunized through subcutaneous or intramuscular injection,and tested using ELISA,ELISpot,and CPE to compare the immune effects of the two injection methods.Results: The pEE12.4 TD-Ab recombinant plasmid was successfully constructed;High expression of TD-Ab was detected by WB in the cell pools;High purity PF-D protein was purified by the construction of a special filler;Specific Ig G binding antibodies were detected in the serum of mice immunized with PF-D protein,and secretion of specific IFN-γ was detected in the splenocytes;Specific Ig G binding and neutralizing antibodies were detected in both subcutaneous and intramuscular immunized mouse serum,and secretion of specific IFN-γ was detected in the splenocytes.Conclusion: Successfully constructed and expressed TD-Ab,and purified high-purity PF-D protein.Through immunization with C57BL/6 mice,it was demonstrated that PF-D protein has good immunogenicity,and the immune effect produced by intramuscular injection is better than that by subcutaneous injection.