Purification et caractérisation d’un antigène somatique et d’une protéase à cystéine excrétée-sécrétée de Fasciola hepatica à intérêt immunodiagnostic.

Abstract

The present study describes in its first experimental part, the isolation of an antigenic molecule from the total homogenate of Fasciola hepatica adult flukes, then purified to homogeneity using gel filtration chromatography followed by a specific affinity chromatography. Among the five antigenic fractions obtained by fractionation of the crude somatic extract on Sephadex G-200 gel, only the first fraction (F.1) gave a highly significant recipitation reaction with the Concanavalin A lectin. It was then purified to homogeneity through a ConA-Sepharose 4B affinity column and characterized by SDS-PAGE electrophoresis as a single-band with apparent MW of about 40 kDa. This pure molecule, whose immunoreactivity was very positive in the presence of rabbit specific antiserum, is therefore endowed with a significant antigenic activity. In addition, the immunodiagnostic reliability of this molecule was verified by Elisa; the assay using the purified fraction as antigen (ELISAPf) proved more sensitive than the Elisa using the excreted-secreted product (ELISAESP) (92.3% of sensitivity for Elisa(Pf) against 72% for Elisa(ESP)). However, the ELISA specificity using the purified fraction was slightly lower compared to that corresponding to excreted-secreted product (93.3% of specificity for ELISA(Pf) against 95.5% for ELISA(ESP)). In the second practical part, a proteolytic activity cysteine-like produced in the supernatant of adult fluke culture medium was purified and characterized by combination of acetone precipitation and two chromatographic methods. Through this three-step purification, the enzyme was purified 11-fold with a specific activity of 1893.9 U/mg and 31.5% recovery. Ultrafiltration was required to increase the purification fold to 13.1 with an overall specific activity of 2268.8 U/mg. The MW of the purified protease was estimated by SDSŔPAGE to be 22 kDa, while the proteolytic activity detection was carried out by zymography on non-denaturing SDSŔPAGE containing the casein as substrate. The enzyme has relative stability over a wide range of pH and temperature with optimal activity at pH 5.5 and 40 °C. Its activity was completely inhibited by specific inhibitors of cysteine proteases, namely 5 mM E-64 and 10 mM iodoacetamide. The practical interest of this purified molecule as an antigen was evaluated by Western blot using sera from sheep experimentally infected with metacercariae of F. hepatica. The pool of sera recognized a single band corresponding to this protease just after the 2nd week of infestation. The early detection of specific antibodies in the sera of infested animals suggests the application of this molecule to the immunological screening of fasciolosis by this technique.