Expression Of Surface Proteins Of Staphylococcus Aureus And Development Of Preliminary Detection Method For The Bacterium

The infections caused by Staphylococcus aureus(S. aureus) have attracted worldwide attention owing to the serious threat to human health and enormous economic losses. S. aureus is an important pathogen that can cause human purulent infection and food contamination. Food can be easily contaminated by this pathogen because of its extensive existence in the environment. In the contaminated food, S. aureus can grow rapidly and produce enterotoxin at the suitable tempreture. Therefore, it is important to develop rapid detection method for S. aureus.In this study, sandwich ELISA detection method specific for S. aureus was developed using surface proteins as targets. This work will be great helpful to the development of rapid detection method for S. aureus in food safety test and clinical diagnosis.In this study, we designed specific primers of iron-regulated surface determinant A (IsdA) gene and serine-aspartate repeat protein D (SdrD) gene, and obtained gene fragments of 801 bp and 2043 bp after PCR amplification with DNA of S. aureus type strains as templates. These gene fragments were ligated into the expression vector pET26b, and then transformed into E. coli BL21(DE3). After being induced by IPTG, the two recombinant proteins were expressed successfully with approximate molecular weight of 38 KDa and 99 KDa, repectively. The expressed proteins were purified with immunoaffinity chromatography and used to immunize New Zealand white rabbits and white Cadillac hens to prepare polyclonal antibodies. Sandwich ELISA for S. aureus was developed using antibody IsdA-IgY as the capture antibody and antibody SdrD-IgG as the detection antibody. The conditions of the ELISA were optimized as follows:the package amount of the capture antibody IsdA-IgY was 2 μg/well; the blocking solution is 0.5% skimmed milk powder in 1×PBS; the detection antibody SdrD-IgG was diluted 500 folds; the enzyme labeled secondary antibody was diluted 10000 folds. For the three type strains ATCC49525, ATCC49521 and ATCC55804, the limit of detection were 1.44×107 cfu/mL,9.14×106 cfu/mL and 7.14×106 cfu/mL, respectively. The limit of quantitation were 5.74×107 cfu/mL, 3.59×107 cfu/mL、3.29×107 cfu/mL respectively. The variations of intra-assay and inter-assay were examined and the results indicated that this method has good stability. Moreover, this method could be well used to detect 16 S. aureus, which indicated its universal applicability for S. aureus detection.