Research On Expression And Immunological Character Of Three-Recombinant Toxins For Staphyloccus Aureus

Food is very important with the manking surviving and developing.The food safety is concerned by the society with the developing in food industry.The food poisoning is the main problem in which the food poisoning bacteria is dominating.Some toxin including SE can cause food poisoning.With the developing of the molecule biotech,the study on food poisoning toxin is increasing and the cognition is enhancing.In the diagnosing and detecting ways,the toxin gene is more credibility and sensitivity sign than others.The detection of poly-recombined toxins may be an innovation which will be more broad-spectrum and quicker.In this experiment we studied S.aureus.we designed and synthesized the primer according the gene sequences which were published in the articles or Genbank.Amplifying the gene fragments which include SEA,SEB and SEE.After purified,the gene fragments were ligated with pUCm-T vector and transformed into E.coli DH5α.The homology was 100% by sequencing.The clond gene fragments were ligated in series with the Linker sequences.We abstained an sole-peptide-chain,in which there were two restriction enzyme sites EcoRI,XhoI.The primers was designed and synthesized that the ending codon was TGA.The full length sequence was amplified through Overlap Extension PCR,then ligated with pET-28a and transformed into E.coli DE3.The homology was 100% that the gene sequence were compared with original sequence. The pET-28a-T were fermented,which induced and expressed by IPTG. The inclusion body of fusion was optimized by SDS-PAGE electrophoresis.Its mass was as same as the predicted molecular weight.Thin-layer scanning is 26.4%.The inclusion body protein was prepared after denaturation the T fusion toxins,and detected the content of protein.We immunized the JiRong Rabbit every 7 day and the total time was 35 days.After immunizing,we collected the blood from the heart and produced the anti-serum and abstracted three natural toxin from S.aureus.The antibodies reacted with the five toxin antigen,which the results were positive but values were different.It proved that the T expressed protein can activate the body producing the antibody.The different bacteria and toxins reacted with antibody specially which the results were not clearly positive by ELISA detection.It proved that the toxic antigen had the better specially.The Sensitivity Test and Agar Diffusion Test are important that there can be used in the detection of toxin in the food poisoning bacteria and construct the board-spectrum,quick,special detecting ways with poly-recombinant toxins.