| Staphylococcus aureus was the predominant pathogen of bovine mastitis and hospital clinical infection, and antibiotic was the main means to treat this infection. However,it was becoming increasingly difficult to control this pathogen due to the enhancing and spreading of bacterial drug resistance caused by long-term, large quantity antibiotics use. In addition, there were still antibiotic residues in milk products by treatment of bovine mastitis with antibiotics which affected food safety. These years,the research on an effective vaccine development was the hot fields to prevent Staphylococcus aureus infection by immunization.The surface polysaccharides (CP5, CP8 and 336PS) were the main antigen components and virulence factors of Staphylococcus aureus, and were also the important target antigens for vaccine development. Because of their small molecular weight and poor immunogenicity, the way to improve their immunogenicity was critical to develop effective vaccine. In this paper, a study would be carried on isolation, purification and carrier protein conjugation of three important Staphylococcus aureus surface polysaccharides and study on immunogenicity of conjugated products as well.Firstly, CP5 and CP8 were isolated from Staphylococcus aureus by autoclaving crushing method, nucleonic acid and protein were removed by different enzymes, and then certain purity CP5 and CP8 were obtained by ion exchange chromatography. For getting the 336PS, the strain was digested by lysostaphin to release polysaccharides, gradient concentration ethanol and different enzymes digestion were used for crude extraction, and further purification by gel filtration chromatography was done to obtain certain purity 336PS.A chemical assay was established to study Staphylococcus aureus surface polysaccharides. The results showed that the purity of CP5, CP8 and 336PS was 67.51%, 63.83%,68.51%,respectively. Purified surface polysaccharides reacted only with anti-homeotype strain serum by double immunodiffusion.A study was done on three surface polysaccharides conjugated antigens preparations and their immunogenicity detection. The purified surface polysaccharides were conjugated to BSA by ADH spacing and EDAC zero-length cross-linking methods. Successful conjugation was identified from determined peak by gel filtration chromatography and ultraviolet scanning.Six surface polysaccharides conjugated products of oil-adjuvant subunit vaccine were prepared to immunize mice. And 3 unconjugated surface polysaccharides, PBS vaccine were prepared for control by the same method. The mice were immunized the vaccine in preimmune,14th days and 28th days,then the serum was isolated and its antibody level was detected by indirect-ELISA.the results showed that the conjugated antigens all could elicit good immune response prepared by both ADH spacing method and EDAC zero-length cross-linking method,but no immunogenicity in surface polysaccharidesTo understand the immune protection, the mice were immunized again in 28th day, and injected the homeotype strain into the outside drawleg after 7 days, clinicopathologic change was observed and scored the clinical index. The challenge results showed that immunized mice with conjugated antigens could be provided better immune protection after homeotype strain challenge.With comprehensive analysis, the conjugated antigens prepared by ADH spacing method were prior to those prepared by ED AC zero-length cross-linking method.To deal with the problem of carrier protein BSA immunized in dairy cow with poor immunogenicity, genetic engineering methods was used to prepare Pseudomonas aeruginosa recombinant exoprotein A. The exotoxin A gene was amplified by PCR to establish a recombinant vector pET-28a-ETA,then transformed into DH5a competent cell. Recombinant vector was extracted and identified by dual-cleavage, PCR and sequencing methods. The objective gene was induced by IPTG, and its product was identified by SDS-PAGE and western blotting, then the optimization of expressed condition, distribution of objective protein was done. A large-scale expressed was then induced by IPTG.The inclusion body was denatured by urea, the best refolding condition and refolding buffer was also optimized. At last, the rEPA was refolded by Ni-NTA on-column refolding method,the results showed that the purity of objective protein was above 93%。The surface polysaccharides-rEPA conjugated antigens were prepared by ADH spacing method. The animal tests showed that the three conjugated antigens had good immunogenicity and could elicit anti-polysaccharides immune response, and the antibody could react with homeotype strain by agglutination test.The research on isolation and purification of CP8 and 336PS, conjugation to carrier protein were the first time to establish this technical methods in China. Mice model and challenge results proved that conjugated antigens had good immunogenicity and immune protection. And the experiment provided technical storage for preparation of Staphylococcus aureus subunit vaccine to bovine mastitis. |
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