| Staphylococcus aureus is a well-known Gram-positive bacterium that is responsible for causing a myriad of infectious diseases. Antibiotics are the main method for controlling the infection by Staphylococcus aureus. Many antibiotics resistant strains emerged for antibiotic abuse. Approximately 40 to 60% of staphylococcal isolates are resistant to multiple antibiotics, including methicillin and vancomycin. And antibiotic-resistant strains are very difficult to treat. Therefore, prophylaxis with a vaccine would be a preferable mode of controlling the debilitating effects caused by this organism.A crucial question in vaccine development is the target population selection for an S. aureus vaccine. There are certain groups that are likely candidates for active immunisation against sta- -phylococcal disease, such as Patients undergoing emergency surgery, residents of nursing hom- -es and other long-term care facilities, men who have sex with men, military personnel, patients undergoing elective surgery, patients with HIV, intravenous drug users, healthcare providers.Every human being possesses antibodies against several S. aureus antigens as a result of S.aureus colonization. Following infection, antibody levels against these antigens (e.g. a-toxin, lipase and teichoic acid) are significantly increased and can be used diagnostically. Despite the presence of these antibodies, we can all get infected and reinfected. Obviously, these antibodies are not protective. Therefore, to produce an effective vaccine, it is necessary to find other antigens that stimulate the production of protective antibodies. However, the virulence of S. aureus is determined by a number of different factors, which makes design of a widely effective vaccine difficult. Various cell components are promising candidates for active and passive immunization strategies such as toxin, capsule, slime, teichoic acids or cell wall bound adhesins. To develop a broadly protective vaccine, we selected twenty proteins of S. aureus as antigens, which has been reported by literature. A combined vaccine with several components may be effective for controlling infection by S. aureus.There are two parts in our research. In the first part, we expressed and purified twenty proteins of Staphylococcus aureus. The genome DNAs of Staphylococcus aureus USA300 and 12598 were extracted and used as the template to amplify the twenty genes by PCR and the acquired fragment was cloned into the pET-21a plasmid. The recombinant plasmids were verified by DNA sequencing, and it turned out that the amino acid sequence encoded by the plasmids were same as expected. The constructs were transformed into E.coli BL21(DE3) respectively. All the target proteins could be expressed at high levels after induced by IPTG. The SP Sepharose F.F column or the Q Sehparose F.F. column according to the character of the proteins was selected for target protein purification. For His tag was attached to C terminal of all the proteins, further purification was conduced by HisTrap column for higher purity. Results showed that we had obtained the twenty proteins with high purity. In the second part, we tested if these proteins could protect BALB/c mice from S.aureus infection. First, we detected the distribution of the aimed genes in the five strains by genome PCR to find genes that expressed more widely in S.aureus isolates. The sera were prepared from the mice immuned with S.aureus and were used to screen the proteins. Then we immunized mice with some of the proteins to determine if they could protect the hosts from S.aureus infection.As so far, we have gotten twenty proteins of S.aureus. According to the finding of PCR and Dot blot, we choose four proteins to detect their effection on S.aureus infection. The study showed that clumping factor A, Fibronectin binding protein A, Fibronectin binding protein B and S.aureus surface protein F couldn,t protect hosts from S.aureus infection, and they may be not the promising candidates for active immunization strategies. |
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