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

SMITH ME; TARGOVNIK AM; CEREZO J; MORALES MA; MIRANDA MV; RODRIGUEZ TALOU J

PROTEIN EXPRESSION AND PURIFICATION

ACADEMIC PRESS INC ELSEVIER SCIENCE

Lugar: Amsterdam; Año: 2016 p. 1 - 38

1046-5928

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