Design of a vector-host system for surface display of heterologous antigens on Bacillus subtilis spores

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

Horco Molle

Congreso; XXXI Jornadas Científicas de la Asociación de Biología de Tucumán; 2014

ABT

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 surfacedisplay system based on the use of bacterial spores, we carried out a transcriptional fusion between the genesenconding a protein of the Bacillus subtilis spore coat, CotB and a immunogenic protein of the casual agent of AmericanTegumentary Leishmaniasis Leishmania (V.) braziliensis, named LbAg3.The structural gene of CotB was amplified fromB. subtilis 168 and subsequently reamplified using specific primers that incorporated a small polylinker toward the 3´ end.The amplicon was cloned in the pRSETa plasmid. The LbAg3 coding region was amplified by PCR from pIVEX-LbAg3and subsequently reamplified adding toward the 3´ end a XmaI site restriction, a coding sequence for six histidineresidues and a BamHI restriction site. This amplicon was cloned directionally downstream of cotB. The obtained fusioncotB-polylinker-lbAg3-6His was digested with enzymes HindIII and BamHI and cloned into the integration vector pDG364obtaining the vector called pSPOK. The efficient surface presentation of the heterologous protein, together with thestability are being evaluated. This vector allows the integration of the genetic construction in the genome of Bacillus andthrough the multiple cloning site will allow working with genes of other antigens.