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

LEONARDO ACUÑA; RAMIRO ORTIZ MOYANO; MIGUEL BASOMBRIO; JORGE MARCO; AUGUSTO BELLOMIO

Horco Molle

Jornada; XXXI Jornadas Científicas de la Asociación de Biología de Tucumán, 2014; 2014

ABT

The safety, stability and easy handling of Bacillus subtilis spores makes this system of particular interest to express in the surface recombinant antigens capable of triggering a protective immune response. In order to obtain a novel surface display system based on the use of bacterial spores, we carried out a transcriptional fusion between the genes enconding a protein of the Bacillus subtilis spore coat, CotB and a immunogenic protein of the casual agent of American Tegumentary Leishmaniasis Leishmania (V.) braziliensis, named LbAg3.The structural gene of CotB was amplified from B. 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-LbAg3 and subsequently reamplified adding toward the 3´ end a XmaI site restriction, a coding sequence for six histidine residues and a BamHI restriction site. This amplicon was cloned directionally downstream of cotB. The obtained fusion cotB-polylinker-lbAg3-6His was digested with enzymes HindIII and BamHI and cloned into the integration vector pDG364 obtaining the vector called pSPOK. The efficient surface presentation of the heterologous protein, together with the stability are being evaluated. This vector allows the integration of the genetic construction in the genome of Bacillus and through the multiple cloning site will allow working with genes of other antigens.