Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10852698 | Biologicals | 2014 | 8 Pages |
Abstract
The beta-haemolysin of Staphylococcus aureus (SA-hlb) is a secreted neutral sphingomyelinase (nSMase) implicated in the pathogenesis of infection and responsible for the characteristic in vitro 'hot-cold' haemolytic ability of the bacterium. Here, we describe the production of a biologically active N-terminally-truncated recombinant SA-hlb protein for use in in vitro assays and as a research tool. Using local isolates of S. aureus, we PCR-amplified an SA-hlb DNA sequence of 891 nucleotides, 99 nucleotides shorter than the full-length molecule, before cloning and sequencing (GenBank accession no. JN580071). The pQE.TriSystem vector (Qiagen, Germany) was used to express recombinant SA-hlb (r-SA-hlb) with a C-terminal 8xHis tag in Escherichia coli JM107 cells. Both JM107 lysate and the purified r-SA-hlb possessed hot-cold lytic activity against sheep and buffalo erythrocytes, which was abolished by incubation at â¥90 °C for 30 min or exposure to dithiothreitol, and could be neutralized by bovine immune sera. Purified r-SA-hlb was also cytotoxic to buffalo mononuclear cells and was effective as a coating antigen for indirect ELISA to screen for reactive sera. Importantly, the r-SA-hlb was suitable for use as a β-toxin in the modified CAMP test. We conclude that the r-SA-hlb protein produced was functionally active and has numerous potential applications.
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Authors
M. Singh, A. Singh, A. Sharma,