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

ACUÑA L; BRACAMONTE E; MOYA A; BARROSO, PA; BELLOMIO A; MARCO, JD

Paraná

Congreso; LIV reunión anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular.; 2018

Sociedad Argentina de Investigación Bioquímica y Biología Molecular.

American tegumentary leishmaniasis (ATL) is an endemic disease in Argentina and there are no vaccines for human application. Heterologous expression of specific antigens in generally recognized as safe bacteria (GRAS) could be a valid alternative for vaccine formulations. In order to obtain a system for protein exposure on Bacillus subtilis, a genetic construction was developed. For that, a transcriptional fusion was created between CotB, a protein expressed in spores, and LbAg1, an immunogenic protein of Leishmania (V.) braziliensis. The structural gene of CotB was 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 re-amplified adding to the 3´ extreme: i) the XmaI restriction site, ii) a sequence encoding for six histidine residues and; iii) the BamHI restriction site. This construction was cloned downstream of cotB. Finally, the hybrid gene obtained cotB-polylinker-lbAg1-6His was digested with HindIII and BamHI and cloned into the integration vector pDG364 obtaining the plasmid called pSPOK. This plasmid may be used for cloning any gene of interest allowing its expression on outer spore surface of B. subtilis. The utility of B. subtilis spores for the delivery and in-vivo LbAg1 presentation is under assay. The safety and easy handling of B. subtilis make this expression system useful for the expression on the surface of bioactive molecules such as recombinant antigens capable of triggering a protective immune response.