INVESTIGADORES
RODRIGUEZ TALOU Julian
congresos y reuniones científicas
Título:
LOW-COST PRODUCTION OF DENGUE VIRUS E-DOMAINIII FOR AN IMMUNOASSAY DEVELOPMENT
Autor/es:
SMITH ME; TARGOVNIK AM; CEREZO J; MIRANDA MV; RODRIGUEZ TALOU J
Lugar:
Panama
Reunión:
Congreso; 5 th Pan American Dengue Reseearch Network Meeting; 2016
Resumen:
Introduction: Dengue is a viral disease transmitted to human by Aedes mosquitoes. The incidenceof dengue has grown dramatically in the last years and around 390 million infections occur each year.Currently, most of the available serological immunoassays use the whole virus as a source ofantigens, what implies high costs, potential biohazard and a high cross reactivity with othersflavivirus. Thus, the aim of this work was to express a recombinant antigen of dengue virus inRachiplusia nu larvae for the development of a specific immunoassay for dengue diagnosis. Therecombinant antigen consists on the domain III of the envelope protein of dengue virus type-2 fusedto hydrophobin (rDomIIIHFB) for its purification by an aqueous two-phase system (ATPS) and for itsimmobilization in immunoassay plates. Materials and methods: Antigen production: Rachiplusia nularvae were infected with a recombinant baculovirus containing the expression cassette forrDomIIIHFB by intrahemocele injection. At 3 day post-infection, larvae were harvested and totalproteins were extracted.Purification: was performed by ATPS with Triton X-114 at 2%, 5% and 8% v/v. Samples of eachstep were analyzed by SDS-PAGE and Western Blot.Protein immobilization: 0.1µg/ml to 12.5µg/ml purified rDomIIIHFB solutions were loaded in multiwellplates (Polystyrene and Polysorp plates, Nunc) and incubated overnight at 4°C. Immunoassay:polystyrene plates coated with rDomIIIHFB were used for the detection of anti-dengue IgG antibodiesin serum samples.Results: R. nu larvae expressed 4.5 mg of rDomIIIHFB per g of larva, with the expected molecularweight of 22 kDa. We could efficiently purified rDomIIIHFB by ATPS. In a single purification step,when using a 2% of surfactant, we recovered 3.3 mg of rDomIIIHFB per g of infected larvae, with apurity of 80% and a yield of 73%. We found that rDomIIIHFB was efficiently immobilized inpolystyrene and Polysorp plates and we selected 6.25µg/ml as the concentration to coat plates forfurther experiments. In these conditions, with the amount of rDomIIIHFB recovered from a single larvawe could coat 22 plates of 96 wells. The immunoassay developed with rDomIIIHFB was able todetect IgG specifics for dengue virus type-2 in patient samples and was not able to detect IgG for theother serotypes. Conclusion:We developed a low-cost expression and purification platform for theproduction of a dengue virus antigen. The rDomIIIHFB-based immunoassay shows specificity fordengue virus type-2 IgG.Funding of research: UBACYT 2014-2017-20020130100298BA, Universidad de Buenos Aires.