Construction of a genetic cassette to express an immunogenic Dengue virus protein in a plant system

MARTÍNEZ CA, GAMARNIK A, GIULIETTI AM AND RODRÍGUEZ TALOU J.

Rosario, Argentina

Congreso; XLII Congreso de SAIB; 2006

Sociedad Argentina de Investigaciones Bioquimicas

  Dengue virus genome encodes three structural (C, prM and E) and seven non-structural proteins, being E the most immunogenic. E protein needs to be directed to the secretory pathway to be glycosilated through a specific signal peptide in the N-terminus. The presence of an ER retention signal in the C-terminus of some proteins increases their stability and the production level of the recombinant protein targeted to the secretory pathway in plant systems. The aim of this work is to obtain a genetic construction with the E protein downstream the rice Glycine-Rich Protein signal peptide (GRP) and upstream the KDEL retention sequence, to express this construction in a plant system and to assay the effect of KDEL sequence on the expression levels of the E protein. A PCR overlapping was designed to make the constructions that resulted in a PCR product containing GRP-E-KDEL and GRP-E with two different restriction sites (Kpn I and Xho I) for its further cloning into to the cassette expression system. We used four different PCRs and five different primers designed specifically to ensamble the constructions correctly. We cloned the genetic constructions in a pGEM T Easy vector for sequencing. They will be cloned in a binary vector under the control of plant promoter and terminator to express them in a plant system and evaluate the levels of E protein for each construction.