Study On The Iron Responsive Surface Determinant B As A Tomponent Of Staphyiococcus Aureus Protein Vaccine

Staphylococcus aureus (Short for S.aureus) is a kind of the Gram-positive bacteria that widely exists in the nature, it has a strong pathogenicity. Different part of the body infected by Staphylococcus aureus will lead to different diseases, such as skin and soft tissue infections, pneumonia, papillitis, bacterial infections, severe patients may even die of the diseases. The iron responsive surface determinant B (Short for IsdB) which plays a very important role in the iron responsive surface determinant system is one of the numerous virulence factors of Staphylococcus aureus. IsdB is very important in Staphylococcus aureus infection and reproduction in the host. At the same time, IsdB is also a novel target for Staphylococcus aureus vaccine research.In this thesis, we maily study the construction of prokaryotic expression plasmid of Staphylococcus aureus natural iron responsive surface determinant B(IsdB) and the truncated IsdB protein(IsdB614, IsdB639, IsdB645), expression, purification, identification, immunogenicity and animal protection experiment. IsdBWT gene sequence is designed and synthesis after the Escherichia coli codon optimization. Then it’s constructed into pET20b-IsdBWT prokaryotic expression plasmid, it’s choosen as the expression plasmid. We use the IsdBWT expression plasmid as template, by designing the truncated gene sequence PCR primers and PCR method to obtain the nucleic acid sequence of IsdB614, IsdB639and IsdB645protein, so as to construct this three truncated protein prokaryotic expression plasmid. The application of E.coli prokaryotic expression system provides a large number of IsdB614and IsdB639protein and some IsdBWT and IsdB645protein, IsdB614and IsdB639protein are separated and purified by the use of affinity chromatography and molecular sieve chromatography. Then all proteins are specificity detection by SDS-PAGE gel electrophoresis and Western Blot identification. The rabbit antibody is used for the identification of immunogenic proteins. The mice intraperitoneal infection model is established for the protein immune protection test, along with the detection of serum antibody titer to detect and evaluate the immune protective effect of this protein.Through this experimental research, we constructed the prokaryotic expression plasmid of IsdBWT, IsdB614, IsdB639and IsdB645successfully, and the two protein—IsdB614and IsdB639were effectively separated and purificated. The purified IsdB639protein showed good immunogenicity in mice, and it also has significant immune protection effect in the attacking experiment. Experimental study of this thesis provide the basis for further development of the effective protein vaccine to inhibite Staphylococcus aureus infection.