Cloning, Expression, Purification And Study On Immunological Effect Of Staphylococcus Aureus Surface Proteins

Staphylococcus aureus causes a number of infections of varying severity incomplicated wound infections which can progress to complicated septicaemia, osteomyelites, septic arthritis or endocarditis. S. aureus is one of the more common bacterial species causing serious hospital-acquired infections. Methicillin-resistant S. aureus (MRSA) strains can resist against most clinically useful antibiotics. As a result of this, vancomycin is often the only effective antibiotic remaining. Moreover, reports from clinical studies have shown the existence of MRSA strains with reduced sensitivity for vancomycin. The advent of Vancomysin-intermediate S. ureus( VISA) strains have great panic on which the infection of S.aureus couldn't cure. This situation has also led to renewed interest in vaccination against S.aureus infections as a complement to antibiotic treatment and prophylactics.Recent research reveals that vaccines based on recombinant staphylococcal extracellar-matrix-binding proteins (ECMBPs) are much more protective.Using PCR technology ,we amplify the fnb and cna genes of S.aureus encode fibronectin binding proteins (FnBP) and collagen binding proteins (Cna). And cloning to the expression vector pET-32a(+) fused with Trx-Tag coding sequence or not, to acquire the recombinant plasmid trx-fnb- pET-32a(+), trx-cna- pET-32a(+),fnb- pET-32a(+) and cna- pET-32a(+). Induction by IPTG, we acquired the high expressed fusion protein Trx-Cna, Trx-FnBP( these proteins account for 58.8%, 52.8% of total cellular proteins) and none fusion protein Cna. In order to get the bivalent protein vaccine . using overlapping primer extension PCR method ,we linked the fnb and cna genes and cloning to pET-32a(+) fused with Trx-Tag coding sequence. The fusion protein Cna-FnBP is highly expressed (account for 26.5% of total cellular proteins).Those proteins was purified by HiTrap Chelating HP and ion exchange chromatography (Q Sepharose HP) respectively. Balb/c and Kunming mice immunized with purified proteins, Trx-Cna, Trx-FnBP, Cna and Cna-fnBP. Antibody titre measured by ELISA. The immunized groups generate a high titre (>108) compared tocontrol group. The presence of immune cells that can be directly stimulated by those antigen was confirmed by measuring splenocyte proliferation in vitro in the presence of the antigen. The result shows those antigens has a good cellular immure response. After the last immunization mice were challenged with S. aureus strain S-6, ( 5 X 107 CFU ,5% MUCIN). The Survival rates amounts to 42.8% (CN group), 50% ( FN group ), 50% (CN-FN group) compare to 25% (Control group) . Analysis by Western blot, the proteins have a good affinity to the antibody sera of immunized animals.Conclusion We get the surface proteins of S. aureus in soluble, which can maintains the high immunogenicity. These plasmids can express a high amount of recombinant proteins. We first linked the fnb and cna genes by overlapping primer extension PCR method to construct bivalent protein vaccine. The vaccine can against S.aureus infections in a more large scale. Experiments in annimals suggest that the proteins can stimulate a good effects on humoral immunity and cellular immunity. To develop the ECMBPs recombinant vaccine has great potential.