Diagnostic potential of Toscana virus N protein expressed in Escherichia coli

Abstract

The nucleocapsid (N) protein of the Toscana (TOS) virus was expressed in Escherichia coli by using a pET15b vector. The recombinant protein was purified by affinity chromatography and was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The recombinant antigen was reactive with positive human sera, and the reactivity correlated very well (r = 0.9) with that of a whole-virus antigen when tested by EIA with 30 TOS virus-positive and 30 TOS virus-negative serum samples. The results demonstrate that the recombinant N protein can be easily produced in a procaryotic system and used for diagnostic assays for TOS virus immunity.

FIG. 1

Scheme for the genetic construct pSDTV-1. The N-protein gene (≃1,800 bp) of TOS virus is inserted at the BamHI site in pET15b (InVitrogen) under the control of the T7 promoter.

FIG. 2

Immunoblot of a human positive serum (lanes a, b, and c) and a negative human serum (lanes e, f, and g) with TOS virus proteins, purified N protein, and recombinant N protein in the sets of lanes, respectively. Low-molecular-mass markers (in base pairs) are indicated on the left (New England Biolabs, Milan, Italy).

FIG. 3

Comparison of the reactivities of the TOS virus IgM-positive sera (A) and IgG-positive sera (B) to the whole virus (□), the purified N protein (), and the recombinant N protein (■) by EIA. The results are expressed as the optical densities (OD) at 450 nm for three independent experiments. Samples 1 to 9 were from patients in the convalescent phase of TOS virus infection, and samples 10 to 30 were from patients in the acute phase of TOS virus infection.