Prokaryotic expression and identification of recombinant cashew nut allergen Ana o 2

Abstract

Objective To express the cashew nut allergen Ana o 2 and identify its immunological activity. Methods Ana o 2 gene was synthesized and inserted into the pET-28a (+) expression vector. The recombinant plasmid pET-28a (+) - Ana o 2 was constructed and sequenced. The correct recombinant plasmid was transformed into Rosetta (DE3), and the recombinant Ana o 2 protein was expressed and purified. The purified recombinant protein was identified by mass spectrometry. The immunological activity of the recombinant Ana o 2 protein was evaluated by enzyme-linked immunosorbent assay (ELISA) and protein immunoblot (Western blot). Results As determined by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the recombinant protein was approximately 54 kD, which was consistent with the theoretical value. The recombinant protein was identified as Ana o 2 by mass spectrometry. ELISA result showed that the level of specific immunoglobulin E (sIgE) in cashew-allergic sera was significantly different from that in negative sera (t=2.44, P<0.05). Western blot result showed that recombinant Ana o 2 had good reactivity with cashew-allergic sera. Conclusion A recombinant expression vector of Ana o 2 had been successfully constructed. Recombinant Ana o 2, which was expressed by prokaryotic system, had good reactivity with cashew-allergic sera.

Keywords

• ana o 2
• gene clone
• recombinant protein
• specific immunoglobulin e
• cashew allergy