Preparation Of Monoclonal Antibodies Against SAG3Protein Of Toxoplasma Gondii And Its Applied Research

The research was conducted to prokaryotic express the SAG3gene containing nosignal peptide of Toxoplasma gondii(T.gondii), and then preparation of the SAG3monoclonal antibody which were used for establishing the double-antibody sandwichELISA.Clone, express and identify the SAG3gene containing no signal peptide ofT.gondii Total RNA was extracted from T. gondii and SAG3gene containing nosignal peptide was amplified by RT-PCR, then sub-cloned into the pET-28a（+）. Therecombinant plasmid pET-28a（+）-SAG3was transformed into E. coli BL21（DE3）and induced with IPTG. The expression of pET-28a-SAG3was induced by IPTG in E.coli BL21（DE3）system; then the fusion protein was identified by SDS-PAGE andWestern blotting. SDS-PAGE result showed that the recombinant protein（Mr38000）was expressed in the form of inclusion body and could specially recognize bypolyclonal antibody against the SAG3of T. gondii.Develop an indirect ELISA which used for detecting SAG3antibodyCheckerboard titration was used to determine the optimal conditions of the indirectELISA. The optimal antigen concentration for coating was0.25μg/hole, and theoptimal dilutions of the sera of T. gondii and enzyme-labeled the second antibodywere1:400and1:3000respectively, blocking agent was the PBST solution of5%nonfat dry milk by chessboard titration. The developed indirect ELISA had no crossreaction with positive serums which mentioned in the following brackets (Neosporacaninum, G. lamblia, C. andersoni, E. tenella and C. parvum). It showed highsensitivity of1:1600and strong specificity.10positive samples and24unknownsamples were detected by the indirect ELISA, the positive rate are100%and20.8%respectively.Preparation and purification of monoclonal antibodies of SAG3proteinRecombinant protein SAG3was purified as immunogen for preparing the monoclonal antibody. Two cell lines (1A1and3E5) were screened by indirect SAG3-ELISA. Bothtiters about1:102400. The immunoglobulin subclass of the1A1and3E5are IgG2aandIgM. Western blotting showed that SAG3protein could specially recognize by1A1and3E5. The light chain and the heavy chain of the antibody could be distinguishedwell by SDS-PAGE means that antibodies were purified successfully.Establishment of double monoclonal antibody sandwich ELISA Purified1A1monoclonal antibody as the capture antibody, HRP-labeled3E5monoclonalantibodies as the detection antibody were used for establishing double monoclonalantibody sandwich ELISA of T. gondii. The optimum conditions established by thesandwich ELISA method was: coated antibody1:100, HRP labeled antibody1:400,test serum dilution of1:10, the best blocking agent for1%BSA. The developedsandwich ELISA had no cross reaction with N. caninum positive serum. The intra andbetween repeated experiments showed that the ELISA has repeatability. The positivesamples and negative samples were detected by this way, the coincidence was100%.85bovine sera samples,63chicken sera samples,58dog sera samples and67pig serasamples were detected by sandwich ELISA, the positive rate are7.06%,12.70%,10.34%and28.36%. The developed sandwich ELISA requires that the seizure samplefor serum, easy to collect, easy to prepare, good specificity, easy to operate,conducive to the promotion and has well application prospects.