ABSTRACT
Background: Staphylococcal protein A [SPA] is a cell wall component of Staphylococcus aureus that binds to different IgG subclasses of human and several animal species. This bacterial protein can be used as an antibody detector in various immunological assays or as an isolation reagent for the purification of antibody molecules via immuno-chromatography procedures
Objectives: Molecular cloning and expression of SPA followed by the purification and conjugation of the recombinant protein to peroxidase enzyme
Material and Methods: Encoding DNA fragment of SPA was amplified and inserted into a prokaryotic plasmid vector for the expression of recombinant SPA fused to a maltose binding protein [MBP]. The recombinant protein was purified using amylose resin column chromatography and conjugated to horseradish peroxidase [HRP] enzyme. Finally, the reactivity of the recombinant SPA was examined against human IgG molecules in ELISA
Results: The results indicated that the recombinant peroxidase-conjugated SPA has a good recognition capacity for human IgG molecules and it was able to produce significant OD values after reacting with human IgG molecules at a concentration up to 0.06 micro g.well[-1]
Conclusions: This recombinant protein can be very useful in all research laboratories and may decrease some of the expenses, e.g. those for preparing conjugated anti-antibodies
ABSTRACT
DNA size markers are widely used to estimate the size of DNA samples on agarose or polyacrylamide gel electrophoresis [PAGE]. DNA markers can be prepared by mixing PCR products with definite sizes. Alternatively, they are prepared by restriction enzyme digestion of the genomic DNA of bacteriophages or natural and synthetic DNA plasmids. The present study describes engineering of a synthetic plasmid which produces a 100 bp DNA ladder, a popular DNA size marker, upon digestion with a single restriction enzyme. Our strategy consisted on sequential cloning of ten PCR products of 100 to 1000 bp in plasmid pTZ57R, using the BamHI and Bg/II restriction sites and releasing the fragments from the recombinant plasmid by enzyme EcoRV. This strategy could be applied to construct various complex synthetic vectors to produce different DNA ladders