Integrated process for the purification and immobilization of the envelope protein domain III of dengue virus type 2 expressed in Rachiplusia nu larvae and its potential application in a diagnostic assay

MARÍA EMILIA SMITH; ALEXANDRA M. TARGOVNIK; JULIETA CEREZO; MARÍA ALEJANDRA MORALES; MARÍA V. MIRANDA; JULIAN RODRIGUES TALOU

PROTEIN EXPRESSION AND PURIFICATION

ACADEMIC PRESS INC ELSEVIER SCIENCE

Lugar: Amsterdam; Año: 2017 vol. 131 p. 76 - 84

1046-5928

Dengue incidence has grown dramatically in the last years, with about 40% of the world26 population at risk of infection. Recently, a vaccine developed by Sanofi Pasteur has been27 registered, but only in a few countries. Moreover, specific antiviral drugs are not available.28 Thus, an efficient and accurate diagnosis is important for disease management. To develop29 a low-cost immunoassay for dengue diagnosis, in the present study we expressed the30 envelope protein domain III of dengue virus type 2 in Rachiplusia nu larvae by infection31 with a recombinant baculovirus. The antigen was expressed as a fusion to hydrophobin I32 (DomIIIHFBI) to easily purify it by an aqueous two-phase system (ATPS) and to33 efficiently immobilize it in immunoassay plates. A high level of recombinant DomIIIHFBI34 was obtained in R. nu, where yields reached 4.5 mg per g of larva. Also, we were able to35 purify DomIIIHFBI by an ATPS with 2% of Triton X-114, reaching a yield of 73% and36 purity higher than 80% in a single purification step. The recombinant DomIIIHFBI was37 efficiently immobilized in hydrophobic surface plates. The immunoassay we developed38 with the immobilized antigen was able to detect IgG specific for dengue virus type 2 in39 serum samples and not for other serotypes.