- 作者：Pooja Jangra ; Ajit Singh<sup> ; sup><sup>adjitprinik@msn.comsup><sup> ; sup><sup>budhadjit59@rediffmail.comsup>
- 关键词：Staphylococcus aureus ; ¦Â-hemolysin neutralization ; Single domain antibodies ; Phage display library ; Indian desert camel
- 刊名：Asian Pacific Journal of Tropical Medicine
- 出版年：2010
- 出版时间：January 2010
- 年：2010
- 卷：3
- 期：1
- 页码：1-7
- 全文大小：436 K
ss=""h4"">Objective
To isolate and characterize Staphylococcus aureus (S. aureus) ¦Â-hemolysin-neutralizing dAbs from phage display library of Indian desert camel.ss=""h4"">Methods
Phage display library of 5¡Á10<sup>7sup> dAb clones of LPS-immunized Indian desert camel constructed in our laboratory was used for selection of S. aureus exotoxin-specific clones by panning technique. Enrichment of Ag-specific clones in successive rounds of panning was assessed by phage-ELISA and phage titration. Different dAb clones binding to S. aureus exotoxin Ags were expressed with C-terminal 6¡Á His tag in E. coli and purified by Ni-chelate chromatography. The expression was verified by SDS-PAGE and western blotting. The purified clones were tested for inhibition of ¡®hot-cold?hemolytic activity in vitro. Resistance to thermal inactivation of the dAb clones was studied by observing the effect of heat treatment from 50 ¡ãC to 99 ¡ãC for 30 mins on the ¡®hot-cold?hemolytic activity in vitro.
ss=""h4"">Results
Several dAb clones binding to S. aureus exotoxins were isolated and enriched by three rounds of panning. The soluble dAb clones were approximately ?6 kDa in size and reacted with 6¡Á His tag specific murine monoclonal antibody in western blot. One of the Ni-chelate affinity purified dAb.6¡Á His clones, inhibited S. aureus ¦Â-hemolysin activity in vitro and resisted thermal inactivation upto 99 ¡ãC.
ss=""h4"">Conclusions
An S. aureus ¦Â-hemolysin-neutralizing dAb clone of possible therapeutic potential has been isolated.