Production and Easy One-Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzym
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ORIGINAL PAPER
Production and Easy One‑Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzymatic Assay (ELISA) S. Ulisse1 · M. Iorio1 · G. Armillotta1 · C. Laguardia1 · L. Testa1 · S. Capista1 · P. Centorame1 · S. Traini1 · A. Serroni1 · F. Monaco1 · M. Caporale1 · M. T. Mercante1 · M. Di Ventura1 Accepted: 13 October 2020 © The Author(s) 2020
Abstract Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (Culicoides spp.) and is caused by Bluetongue virus (BTV). BTV is the type species of the Orbivirus genus within the Reoviridae family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay. Keywords BTV · Recombinant VP7 · Baculovirus · Supernatant · Affinity chromatography · ELISA
Introduction Bluetongue virus (BTV) is a member of the Orbivirus genus of the family Reoviridae [1, 2] and infects sheep, cattle and wild ruminants. BT has evolved in the past ten years from an exotic disease, restricted to warm Sub-Saharian climates,
to a more widespread disease, with the potential of becoming endemic in new areas with temperate climates [3, 4], causing high economic impact on the international livestock industry. BTV has a double stranded RNA (dsRNA) genome formed by 10 segments encoding 7 structural and 4 non-structural proteins [5–8]. The BTV virion consists of
* M. Iorio [email protected]
A. Serroni [email protected]
S. Ulisse [email protected]
F. Monaco [email protected]
G. Armillotta [email protected]
M. Caporale [email protected]
C. Laguardia [email protected]
M. T. Mercante [email protected]
L. Testa [email protected]
M. Di Ventura [email protected]
S. Capista [email protected]
1
P. Centorame [email protected]
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Teramo, Italy
S. Traini [email protected]
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a triple-layered icosahedral protein capsid [7, 9], that gives the name to the species. The outer capsid layer of the virion is formed by VP2 and VP5 that together elicit virus neutralizing antibodies [10]. To date, at least 27 serotypes have been identified worldwide [11–14]. The viral internal core is composed of two layers, constituted by VP3 (sub core) and VP7 (core surface layer) [6, 7] the
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