الفهرس | Only 14 pages are availabe for public view |
Abstract The aim of this work is to study the role of some virulence factors produced by S. aureus (CHIPS, SCIN and SpA) in inhibiting the innate immune system. To achieve this goal, recombinant DNA technology was applied to construct bacterial expression systems of CHIPS, SCIN, and SpA. The SDS-PAGE analysis showed that CHIPS was highly expressed in pET-28 a+ expression vector after 18 hours of induction in a soluble form. SDS-PAGE of SCIN showed that it is best expressed in pRSET-B vector after 4 hours of induction and it was also present in the form of soluble protein. The expressed proteins with 6xHis tag were purified on a Ni+2 Sepharose 6 Fast Flow packed column. The purified recombinant CHIPS was then used to assess the effect of CHIPS on neutrophil phagocytosis by incubating opsonized S. aureus with human PMNs in presence and absence of recombinant CHIPS. Samples were taken at different time points and the viable bacteria were counted. Results showed a significant increase in the viable count of S. aureus in presence of recombinant CHIPS when compared to control experiment indicating its inhibitory effect on phagocytosis. On evaluation of the effect of recombinant SCIN and CHIPS on complement activation through lectin and alternative pathways using ELISA, the recombinant SCIN significantly decreased the levels of C3b deposition from human serum via the two examined pathways while recombinant CHIPS did not show any significant inhibition. The effect of recombinant SCIN and CHIPS on complement activation through lectin pathway was also determined in-vivo by using three groups of BALB/c mice each contained three mice. Two groups were injected intra-peritoneally with either of the recombinant proteins and the control group was injected with BSA. Then blood samples were taken after 6 and 12 hrs and serum was separated to assay the C3b deposition on mannan coated ELISA. Recombinant SCIN showed significant decrease in C3b deposition when compared to the control serum. |