Abstract

LEMOS, Gilda et al. Expression and purification of a full-length recombinant NS1 protein from a dengue 2 serotype viral isolate. Biotecnol Apl [online]. 2013, vol.30, n.3, pp.187-193. ISSN 1027-2852.

Dengue is an emerging disease that poses a threat to one-third of the global human population and produces over 50 million reported cases in tropical and sub-tropical regions every year. An accurate diagnosis of dengue infection is essential for timely management of the disease. NS1 is a 46- to 50-kilodalton highly conserved dengue virus glycoprotein that can be detected during the febrile phase of dengue virus (DENV) infection in both primary and secondary cases. This protein is a specific marker of DENV infection, and a sensitive test for NS1 would, if used together with IgM detection, provide an excellent diagnostic approach. Although the NS1 protein can be isolated from mammalian cell tissue cultures infected with DENV, this procedure is unsafe, laborious, and expensive and has very low yields, making it unsuitable for a large amount of antigen production. In this work, and with the objective of carrying out immunization experiments in mice, we cloned the full-length NS1 region from DENV serotype 2 (rNS1) in the vector pET28a with a 6xHis tag at the N-terminus. The protein was expressed in the Escherichia coli strain Rosetta as inclusion bodies, at the expected size of approximately 46 kDa, and further purified by metal-chelating affinity chromatography (IMAC) under denaturing conditions. Human sera from dengue positive cases showed reactivity to the recombinant NS1 protein by ELISA and Western blot. The unfolded rNS1 was directly used as immunogen. The polyclonal antibodies elicited in immunized mice with the recombinant antigen recognized the natural NS1 antigen from serotype 1 (sNS1).

Keywords : dengue virus; NS1; diagnosis; recombinant protein.

        · abstract in Spanish     · text in English     · English ( pdf )