A genetic system to express recombinant antigens in Bacillus for leishmaniasis vaccines development

ACUÑA LEONARDO; BRACAMONTE MARIA ESTEFANIA; AGUSTIN MOYA ALVAREZ; BARROSO PAOLA ANDREA; BELLOMIO AUGUSTO; MARCO JORGE DIEGO

Parana, Ente Rios

Congreso; 54th Annual Meeting Agentine Society for Biochemistry and Molecular Biology LIV Reunion Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2018

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

American tegumentary leishmaniasis (ATL) is an endemic disease in Argentina and there are no vaccines for human application. Heterologousexpression of specific antigens in generally recognized as safe bacteria (GRAS) could be a valid alternative for vaccine formulations. In order toobtain a system for protein exposure on Bacillus subtilis, a genetic construction was developed. For that, a transcriptional fusion was createdbetween CotB, a protein expressed in spores, and LbAg1, an immunogenic protein of Leishmania (V.) braziliensis. The structural gene of CotBwas amplified by PCR from B. subtilis 168, subsequently reamplified using specific primers to incorporate a multiple cloning site towards the 3'extreme and cloned into the pRSETa plasmid. Structural gene encoding LbAg1 was amplified from L. (V.) braziliensis and subsequently reamplifiedadding to the 3' extreme: i) the XmaI restriction site, ii) a sequence encoding for six histidine residues and; iii) the BamHI restrictionsite. This construction was cloned downstream of cotB. Finally, the hybrid gene obtained cotB-polylinker-lbAg1-6His was digested with HindIIIand BamHI and cloned into the integration vector pDG364 obtaining the plasmid called pSPOK. This plasmid may be used for cloning any geneof interest allowing its expression on outer spore surface of B. subtilis. The utility of B. subtilis spores for the delivery and in-vivo LbAg1presentation is under assay. The safety and easy handling of B. subtilis make this expression system useful for the expression on the surface ofbioactive molecules such as recombinant antigens capable of triggering a protective immune response.