Leishmania major: production of recombinant gp63, its antigenicity and immunogenicity in mice.
The Mr 63,000 membrane polypeptide (gp63) is one of the Leishmania receptors for host macrophages and has been shown to protect mice from infection. The gene encoding gp63, the major Mr 63,000 surface glycoprotein of L. major promastigotes, has been expressed as a fusion protein with the enzyme glutathione S- transferase encoded by the parasitic helminth Schistosoma japonicum. This fusion protein was recognized by polyclonal antibodies to the native Leishmania gp63 polypeptide. The insoluble gp63 fusion protein was purified by SDS-PAGE and electroelution and was used to raise antibodies in rabbits. These rabbit anti-gp63 antibodies recognized the fusion protein and the denatured parasite gp63 on immunoblots and by immunofluorescence on fixed promastigotes, but did not recognize the native molecule on live organisms. However, antibodies raised against native promastigote glycoproteins, affinity purified on solid-phase gp63 fusion protein, recognized both native and denatured gp63, suggesting the presence of native determinants in the recombinant protein. The gp63 fusion protein did not protect mice of either healer or nonhealer phenotype from challenge infection with live promatigotes. The implications of these results for the engineering of recombinant DNA-produced molecular vaccines are discussed.
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Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
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McMaster
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D000818:Animals; D000953:Antigens, Protozoan / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D004195:Disease Models, Animal; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007895:Leishmania tropica / Q000276:immunology*; D007896:Leishmaniasis / Q000517:prevention & control*; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D008666:Metalloendopeptidases; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D011233:Precipitin Tests; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D014611:Vaccination; D014614:Vaccines, Synthetic
D000953:Antigens, Protozoan; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania