| Transmissible gastroenteritis(TGE)is a highly exposed intestinal infectious disease caused by swine transmissible gastroenteritis virus,(TGEV)in pigs and infects pigs of all ages and breeds,especially to the 10 days old piglets,with a fatality rate of up to 100%,which has brought significant losses to pig industry around the world.TGEV is taxonomically classified into the coronavirus genus within the coronavirus family.The(Spike Protein)S protein on the surface of the coronavirus s a key protein for the virus invading cells and can bind angiotensin converting enzyme2(ACE2),causing conformational changes in S protein and promoting virus infection and invasion.Some studies also demonstrated that the development of COVID-19 antibody drugs based on SARS-COV-2 RBD as a target is effective for preventing virus infection.Therefore,this proposal aims to prepare monoclonal antibodies to the RBD protein of TGEV and investigate its effect on viral TGEV infection.We ultimately try to establish an in vitro TGEV detection method using his antibody.Part1,preparation and validation of monoclonal antibodies against TGEV RBD protein.By synthesizing the TGEV RBD gene,the recombinant prokaryotic expression plasmid pET28a-RBD was constructed and the protein expression and purification were performed.Purified RBD proteins were immunized to BALB/c mice.After three immunizations,immunological splenocytes and myeloma cells were harvested for cell fusion according to conventional methods,and then one positive hybridoma cell line 2C2 with stable secretion of anti-RBD antibody was obtained by indirect ELISA screening.Finally,ascites induced in mice was used to obtain sufficient amount of monoclonal antibodies.The antibody characterization results showed that 2C2 is the IgGl isotype;the McAb specifically recognizes TGEV in cells;and the ascites antibody titer is 100 ×212.To further determine the 2C2 antigen recognition epitope,RBD clones were constructed by truncation.The results show that:the 2C2 recognition epitope is 708aa～740aa.Part 2,the preparation and identification of polyclonal antibodiesRabbits were immunized with RBD protein,rabbit poly anti sera were obtained and the polyclonal antibodies were purified by Protein G column purification.The antibody titer measured by the ndirect ELISA method was 100 × 28.Both Western-Blot and IFA assays showed that polyclonal antibodies can bind specifically to TGEV.Part 3,the establishment of the double-antibody sandwich ELISA method.The McAb and polyclonal antibody we prepared were paired,and the results showed that 2C2 McAb was capture antibody,RBD protein polyclonal antibody was the best as a detection antibody.Then detectio conditions were optimized:coated concentration of McAb was 2μg/ml,polyclonal antibody dilution of 1.56μg/ml,optimal blocking solution of 5%BSA,and optimal color development time of 37℃ for 10min,respectively.The established method was tested by sensitivity and specificity,which indicated that the method can specifically capture the TGEV.Conclusion:(1)in this study,we prepared monoclonal antibody against TGEV RBD protein,identified its titer,purity,subclass,epitope,and verified the effect of monoclonal antibody on TGEV replication;(2)RBD polyclonal antibody was prepared and its purity and titer were identified;(3)a sandwich ELISA method with monoclonal antibody as capture antibody and polyclonal antibody for detection antibody was established. |
