Abstract

In this this study, a cloning plasmid named pMD19-FnBPB-ClfA was constructed, by PCR specific amplifications of genes from region D, fibronectin-binding protein B (FnBPB) and region A in clumping factor A (ClfA), all from Staphylococcus aureus. Splicing by overlap extension using PCR tandem gene FnBPB-ClfA was performed and then gene segments of pMD19-FnBPB-ClfA were inserted into a prokaryotic expression vector named pET-32a (+). They were ultimately transferred into the host strain BL21 (DE3), resulting in the expression plasmid pET-FnBPB-ClfA. SDS-PAGE demonstrated an extrinsic protein belt consistent with the desired protein at 51 kDa, when the expression constructed was induced with 1 mmol/L isopropyl β-D-1-Thiogalactopyranoside (IPTG). Western-blot identification demonstrated consensus between the expressed protein and the endogenous protein. After purification and emulsification with Freund's adjuvant, the expressed protein was used to immunize mice. After three subsequent immunizations in the same mice, we gained a highly effective antiserum. Through ELISA, tube agglutination, phagocytosis of opsonized experiment, and antibodies against S. aureus adhesion ability test, it was demonstrated that the fusion gene was successfully expressed in prokaryotic cells, the expressed protein was adherence active, the prepared immune antiserum was capable of preventing S. aureus from adhering to bovine fibrinogens and the antiserum had functions of phagocytosis and opsonization.

Key words: Staphylococcus aureus, FnBPB, ClfA, splicing by overlap extension polymerase chain reaction (PCR), prokaryotic expression.