Studies On Preparation Of Staphy-Lococcal Enterotoxin B Protein Vaccine And Its Immune Effect

Staphylococcus aureus is responsible for causing a myriad of infectious diseases that is a well-known Gram-positive bacterium. It has developed resistance to multiple drugs for antibiotic abuse, especially the rapid spread of methicillin-resistant strains. And antibiotic-resistant strains are very difficult to treat. Therefore, prophylaxis with a vaccine would be a preferable mode to comdat against the pathogen.We mutated three points according to the spatial structure of SEB, then optimized the codon and expressed by prokaryotic expression system and finished the immune protection in Balb/c mice after purifying. The results were as followings.(1) We obtained gene sequence of SEB that had been mutated three points according to its spatial structure after codon optimization and sequence synthesis.(2) The pET-22b expression vector was chosen, and Nde I and Hind III restriction sites were selected. The prokaryotic expression vector was confirmed to be constructed successfully by restriction enzyme digestion, PCR and sequencing.(3) The expression condition was optimized in order to facilitate purification and promote the expression of soluble fusion protein. There was a lot of soluble fusion protein expressed in the triple-checked condition that IPTG was 1mmol/L, induced by 8h in 37℃. The concentration of fusion protein was 10mg/mL after purified by ion-exchange column and quantitied by BCA method and condensed by dialysis bag.(4) Female Balb/c mice were immunized with the rSEB protein and bleed in first week after immunization. There were two groups, one is injection group plus Al(OH)₃ adjuvant and the other is nasal group plus quarter of ammoniated gel adjuvant. Sera were separated for ELISA testing and evaluation of antibody levels of rSEB protein. The mice in injection group were challenged with wide-type SEB toxin after twice immunization, and the mice in nasal group were challenged with wide-type SEB toxin after three times immunization. The liver, spleen, lung, kidney of these mice were taken for histopathological observation so as to evaluate the protective effect of rSEB in Balb/c mice. The results showed that the rSEB protein produced a higher antibody levels and better protective effects whether the mice were immunized by injection means or nasal approach.