Production And Primary Application Of Monoclonal Antibodies Against Recombinant Spike Protein Of Tgev

Recombinant S protein of porcine transmissible gastroenteritis virus (TGEV) was efficiently expressed in E.coli in this research, SDS-PAGE analysis showed that the recombinant protein existed as inclusion body in the E.coli. Inclusion body was purified through washing them step-by-step, The target proteins was isolated by the method of cutting the gel slices, electroeluting, refolding and concentrating, and this protein was obtained by this method, which was antigen for immunization. The result indicated 8M urea could dissolve inclusion bodies after dissolved inclusion bodies by different concentration of urea and guanidine hydrochloride. The target protein was purified by Ni-NAT agarose affinity column. After refolding the target protein by the method of dilution, one step, step-by-step, the result indicated that refolding protein by step-by-step was the best way, and this protein was obtained for the detection of antigen protein. Western blot analysis showed that the purified protein had specificity of the response. Porcine transmissible gastroenteritis virus was cultured on PK-15 cells, after concentrated and purified, the antigen for detection was aquired.When recombinant TGEV S protein was used as antigen for detection, the optimum work conditions for indirect enzyme-linked immunosorbent assay (indirect-ELISA) were developed as follows, the concentration of coating antigen was 4μg/mL,the coating fluid is CBS (pH9.6, 0.05M), the time of coating was stored at 4℃overnight ,the blocking material was 1% gelatin, the dilution of the labeled goat-anti-mouse enzyme was 1:1000, the role of time substrate for 15min. TGEV antigen for the detection, the concentration of coating antigen was 1:1600, the coating fluid is CBS (pH9.6, 0.05M), the time of coating was1 hour at 37℃and then stored at 4℃overnight, the blocking material was 1% gelatin, the dilution of the labeled goat-anti-mouse enzyme was 1:1000, the time of substrate reaction for 15min.Three hybridoma cell strains secreting monoclonal antibodies (McAb) against TGEV was developed by cell fusion after immunizing the Balb/c mice with purified recombinant TGEV S protein. Three hybridoma cell strains named 1G6, 2F8 and 3E5 were obtained by indirecenzyme-linked immunosorbent assay method. These hybridoma cell strains can stably secrete McAbs after twenty-five serial passages and freezing-thawing four times in six months. The ELISA titer of antibodies against recombinant TGEV S protein in culture supernatant were 1:3200, 1:6400, 1:6400 and in ascites were 1:5×105, 1:5×106, 1:5×106 for strains 1G6, 2F8 and 3E5. Respectively, pathogen detection of indirect ELISA shows the McAb has no cross-reaction with the classical swine fever virus (CSFV), porcine circovirus type 2 virus (PCV2), porcine parvovirus (PPV) no cross-reaction. Western blot analysis showed that the antibodies can specifically bind with recombinant TGEV S protein.Antigen capture enzyme-linked immunosorbent assay was developed by the McAbs in ascite of the cell strains, and there was no significant difference repeated this method after four times, these show that the double Sandwich ELISA Indirect detection methods have good stability. All the experimental results show that the McAbs against recombinant TGEV S protein were highly specific to detect pathogen, which laid the foundation for rapid and accurate monitoring of pathogen diagnosis and differential diagnosis of the TGEV.