Purification, characterization, and immunolocalization of paramyosin from the adult stage of Fasciola hepatica

Paramyosin, a vaccine candidate in different helminthiases, was purified from the adult liver fluke Fasciola hepatica using two different procedures. The first started with a crude extraction of paramyosin in high-salt buffer followed by gel filtration chromatography and two precipitation-solubilization cycles; in the second, anion exchange chromatography replaced the gel filtration step. In both cases, the apparent molecular weight of the purified protein determined by sodium dodecyl sulfate gel electrophoresis under reducing and non-reducing conditions was 97 kDa and 200 kDa, respectively. The molecular weights were consistent with the presence of a dimeric protein linked by disulfide bridges. Western blot analysis showed that the dimeric and monomeric forms were both recognized by an antiserum raised against the F. hepatica 97 kDa band (alpha-FhPmy), and by an anti-Schistosoma mansoni paramyosin immune serum. Immunohistochemistry using alpha-FhPmy demonstrated the localization of paramyosin within the subtegumental muscle and in muscle cells surrounding the gut of adult parasites. We also observed labeling of extramuscular structures like testes, surface lamellae of the gut and the tegument of adult flukes.