Cloning and expression of a rotavirus VP6-Flic131 fusion protein

D. SILVESTRE; M.H.ARGUELLES; M.G.MANDILE; GRISELDA MORENO; G.GLIKMAN; A.CASTELLO; M.RUMBO; F.TEMPRANA

Córdoba

Congreso; LII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2016

SAIB

Group A rotavirus is the major etiologic agent of acute gastroenteritis in children. Although, mortality rates have been reduced since the implementation of attenuated vaccines, research on alternative vaccination strategies continues, particularly focused on those based in non-living pathogens. Specifically, our interest is the evaluation of needle-free alternatives able to induce an immune response after mucosal administration. Among rotavirus, VP6 is the most immunogenic and conserved protein. On the other hand, flagellin has been proposed as an important mucosal adjuvant. Thus, fusion ORF containing FliC131, a deletion mutant of S. enterica flagellin, and a fragment of VP6 protein, BB (aminoacids 143 to 334), was constructed by SOE-PCR, cloned and expressed in E. coli. Protein expression was analyzed by SDS-PAGE and Western blot. Results showed that FliC131-VP6BB was efficiently expressed and recognized by specific antibodies against rotavirus and flagellin, although small amount of FliC131-VP6BB was found in the soluble protein fraction. The biological activity of this fusion protein was assessed on Caco-luc reporter cell showing that it stimulates a dependent pathway activation of TLR5. Protein purification will be performed in order to evaluate its immunogenic potential for mucosal administration to mice in a rotavirus model.