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Leishmania major: production of recombinant gp63, its antigenicity and immunogenicity in mice
E Handman1, L L Button, R W McMaster
1Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Abstract:
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.
Insights
Recombinant Leishmania gp63 fusion protein, a potential vaccine candidate, was created but did not protect mice from infection. Further research is needed to engineer effective molecular vaccines against Leishmania.
Area of Science:
- Immunology
- Molecular Biology
- Parasitology
Background:
- Leishmania gp63 is a major surface glycoprotein involved in macrophage interaction.
- gp63 has shown potential in protecting against Leishmania infection in mice.
- Developing effective vaccines against Leishmania remains a significant challenge.
Purpose of the Study:
- To express the Leishmania gp63 gene as a fusion protein with glutathione S-transferase.
- To generate antibodies against the recombinant gp63 fusion protein.
- To evaluate the protective efficacy of the gp63 fusion protein as a molecular vaccine candidate.
Main Methods:
- Gene expression of L. major gp63 fused to Schistosoma japonicum glutathione S-transferase.
- Purification of the insoluble gp63 fusion protein using SDS-PAGE and electroelution.
- Antibody generation in rabbits and characterization using immunoblots and immunofluorescence.
Main Results:
- The fusion protein was recognized by antibodies to native gp63.
- Rabbit anti-gp63 antibodies recognized denatured gp63 but not the native molecule on live parasites.
- Antibodies against native glycoproteins, purified using the fusion protein, recognized both native and denatured gp63.
- The gp63 fusion protein failed to protect mice against Leishmania challenge.
Conclusions:
- The recombinant gp63 fusion protein contains native determinants but does not confer protection.
- Further optimization is required for developing effective recombinant DNA-based molecular vaccines against Leishmania.
- Understanding gp63's role in host-parasite interactions is crucial for vaccine design.