Production and Easy One-Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzymatic Assay (ELISA)

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.

Development of a Competitive Enzyme-Linked Immunosorbent Assay Based on Purified Recombinant Viral Protein 7 for Serological Diagnosis of Epizootic Haemorrhagic Disease in Camels

“…The use of an automated system to purify recVP7 through affinity chromatography offered several advantages, speeding up the process, and its standardization [ 39 – 42 ]. We tested several conditions, and the highest quantity of purified recVP7 was obtained from the supernatant; indeed, the amount of VP7 purified from the supernatant was 15 mg, in contrast to 1 mg obtained from the pellet, even if the protein purity level of sVP7 was lower than the purity of pVP7, as already occurred in previous studies [ 33 ].…”

“…Sf9 pellet treated as previously described and Sf9 supernatant both containing the EHDV recombinant VP7 were subjected to IMAC, according to the method described by Ulisse et al [ 33 ].…”

Improved Efficiency of Bluetongue Viral Antigen Isolation for Successful Immunization

“…The effectiveness of this alternative diagnostic method has been proven in the work. In a study (Ulisse et al 2021), 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. Thanks to the use of the supernatant, the authors obtained a high quantity of recombinant protein with high purity level by an easy one-step procedure.…”