Evaluation of the potential of Mycobacterium smegmatis as vaccine Candidate against tuberculosis by in silico and in vivo studies

In this study, we scanned multiple published databases of gene expression in vivo of M. tuberculosis at different phases of infection in animals and humans, to select 38 proteins that are highly expressed in the active, latent and reactivation phases. The selected proteins were predicted for T and B epitopes. For each proteins, the regions containing T and B epitopes were selected at the same time to look for identical epitopes on M. smegmatis based on sequence alignments. Preliminary studies of humoral immunogenicity and cross-reactivity with M. tuberculosis in mice using two M. smegmatis-derived experimental vaccines were carried out, demonstrating the immunogenicity of M. smegmatis proteoliposomes and the recognition of M. tuberculosis proteins by the sera of animals immunized with this vaccine candidate. The conjunction of in silico and in vivo studies suggested the potential for future evaluation of M. smegmatis as vaccine candidate against tuberculosis using different strategie

Strategy for the purification of soluble fibronectin from human plasma, as ligand for affinity chromatography

El presente trabajo se desarrolló con el objetivo de purificar y caracterizar fibronectina soluble procedente de plasma para su posterior aplicación en la purificación de proteínas de adhesión. Se inmovilizó gelatina en Sefarosa 2BCL como soporte cromatográfico para la preparación de una columna. Se estudiaron diferentes alternativas de elución con arginina 0.5 M y 1M, con glicina -NaCl, acetato de sodio - NaCl y urea - NaCl, lo cual confirmó las ventajas en el uso de la arginina como agente eluyente. Se incorporó una segunda etapa de purificación en una columna de Heparina - Sefarosa 4B CNBr a partir de la que se obtuvo fibronectina con alto grado de pureza en estado nativo, comprobado por SDS-PAGE. La proteína purificada se inmovilizó en una matriz de Sefarosa con un grado de sustitución de 0,98 mg fibronectina/mL de gel lo cual permitió una capacidad de adsorción de gelatina libre con una recuperación de 8 mg (0,64 mg gelatina/mL de gel). La utilización de esta matriz permitirá su aplicación preliminar en la purificación de proteínas de adhesión que podrían ser usadas como antígenos en vacunas contra leptospirosis.Soluble fibronectin from human plasma was purified and characterized in order to be applied in the purification of adhesion protein. Gelatin was immobilized in a chromatographic bed as Sepharose 2B CL to prepare a column. Different alternatives of elution with 0.5 M and 1 M arginin, glicin- NaCl, sodium acetate - NaCl and urea - NaCl buffers, confirmed the advantages of the arginin as elution solution in this column. Second step of purification in a column of Heparin - Sepharose 4B CNBr was included to obtain the native fibronectin with high purity degree, verified by SDS-PAGE. Purified protein was immobilized in a matrix of Sepharose with a substitution grade of 0.98 mg fibronectin/mL of gel that permited an adsorption capacity of free gelatin to the column of 8 mg (0.64 mg gelatin/mL of gel). This matrix will allow its preliminary application in the purification of adhesion proteins which could be used as antigens in vaccines againt leptospirosis.Colegio de Farmacéuticos de la Provincia de Buenos Aire