Virology & Mycology
Open Access
ISSN: 2161-0517
+44 1223 790975
ISSN: 2161-0517
+44 1223 790975
Ana Carolina Viegas Carmo1,2,4, Dalton Nogueira da Silva Giovanni3,4, Mariana Tonelotto4, Nathalia Delazeri de Carvalho4, Karina Senna Villar4, Fernando Luiz Kamitani4, Kamilla Alves Carvalho5, Erika Ohta Watanabe5, Fabiana Regina Xavier Batista5, Darci Moraes Barros-Battesti6 and Ronaldo Zucatell
Posters-Accepted Abstracts: Virol-mycol
Insect hemolymph studies demonstrated the presence of active principles such as antiviral. The aim of this work was cloning, expression and concentration of the recAVLOEc protein with antiviral activity from L. obliqua hemolymph. For that, a PCR with specific primers for the antiviral protein, based on the sequence previously cloned in bac-to-bac system, was made. Restriction sites were inserted in the primer for connection to the plasmid pET28a. First reaction was performed with the restriction enzyme BamHI, followed by digestion with the enzyme HindIII. After binding insert in the vector, the construction was selected in E. coli BL21 (DE3) pLysS and the cloning was confirmed by sequencing. The protein expression was induced with IPTG (1mM) at 0.7 DO at 37�C for 4h. In order to concentrate the target-protein, precipitation by salting-out method was used. Initially, ultrasound bacterial lysis was performed. After centrifugation (8,000 rpm x 10 min), the saturated salt solution (sodium or ammonium sulfate) was added to the supernatant phase of cellular lysate. This solution was maintained for 12h at 4�C until the formation of the precipitate phase. The solubility curve of recAVLOEc was determined by measuring the protein composition of the supernatant phase. The effectiveness of salts as precipitant agents was verified. In addition, antiviral tests were performed and a titration was made with Measles virus infecting VERO cells, in which 24 μg recAVLOEc/mL resulted in the viral title 256-fold lower than control.