Diseño de un sistema vector-hospedador para la expresión de antígenos heterólogos en esporas de Bacillus subtilis

ACUÑA L; ORTIZ MOYANO R; BELLOMIO A; ACUÑA L; BASOMBRÍO MA ; MARCO JD2

BIOCELL

INST HISTOL EMBRIOL-CONICET

Lugar: Mendoza; Año: 2014 vol. 38

0327-9545

The safety, stability and easy handling of Bacillus subtilis spores makes this system of particular interest to express in thesurface recombinant antigens capable of triggering a protective immune response. In order to obtain a novel surface displaysystem based on the use of bacterial spores, we carried out a transcriptional fusion between the genes enconding a protein ofthe Bacillus subtilis spore coat, CotB and a immunogenic protein of the casual agent of American TegumentaryLeishmaniasis Leishmania (V.) braziliensis, named LbAg3.The structural gene of CotB was amplified from B. subtilis 168and subsequently reamplified using specific primers that incorporated a small polylinker toward the 3´ end. The ampliconwas cloned in the pRSETa plasmid. The LbAg3 coding region was amplified by PCR from pIVEX-LbAg3 and subsequentlyreamplified adding toward the 3´ end a XmaI site restriction, a coding sequence for six histidine residues and a BamHIrestriction site. This amplicon was cloned directionally downstream of cotB. The obtained fusion cotB-polylinker-lbAg3-6Hiswas digested with enzymes HindIII and BamHI and cloned into the integration vector pDG364 obtaining the vector calledpSPOK. The efficient surface presentation of the heterologous protein, together with the stability are being evaluated. Thisvector allows the integration of the genetic construction in the genome of Bacillus and through the multiple cloning site willallow working with genes of other antigens.