Fig 1. Cloning, expression, and purification of rNS1.

A) Schematic representation of expression constructs pcDNA3.1-sec-NS1-6xHis (upper) and pcDNA3.1-sec-NS1-V5 (lower). Sec corresponds to an immunoglobulin leader sequence at the N-term; rNS1, recombinant nonstructural protein 1; 6xHis and V5 are tags cloned at the C-term for protein purification and mice immunization, respectively. Reference strains are also indicated. B) Coomassie blue staining of purified proteins: 2 μg of purified NS1 proteins were loaded for each virus. C) Western blot analysis of purified rNS1. Left panel, WB of purified rNS1 proteins diluted in reducing Laemmli sample buffer (LB) and denatured by boiling using an anti-6xHis-tag mAb (WB: H6). Middle panel, WB of rNS1 performed in non-denaturing/non-reducing conditions (without heating the proteins and LB without 2-βME). Right panel, WB of purified rNS1 proteins analyzed by western blot under native conditions (without heating the proteins; without 2-βME and without SDS in the LB buffer, gel, and running buffer). rNS1 monomers (mon), dimers (dim), and hexamers (hex) are indicated. D) Endoglycosidase analysis of purified rNS1. All rNS1 proteins were treated (+)/or not (-) with PNGase F and/or Endo Hf enzymes for 1.5 h at 37 °C. WB analysis was assessed by 10% SDS-PAGE under standard conditions using an anti-6xHis-tag mAb (WB: H6).