Name: Christensen, Claus Bo Vöge Home Country: Denmark Research Country: Project Period: 1994-1997   Title Cloning, isolation and characterization of four immuno-reactive protein antigens from axenically grown Leishmania donovani amastigotes Abstract The T cell blotting method was investigated for use as a relevant screening assay for T cell antigens. By the use of tetanus toxoid as model antigen, properties inherent to the suggested and used acrylamide separation matrix was found to lower or inhibit T cell responses. The problems encountered could, however, be overcome by changing the acrylamide separation matrix to an agarose separation matrix. The agarose matrix although an alternative, was not as efficient in separating antigen below 20 kDa. This and the continuous demand for T cells and antigens for separation in the T cell blot assay lead to development of an alternative approach to identify antigens from Leishmania donovani. Protocols for culturing axenic Leishmania donovani promastigotes and amastigotes were used to generate both protein antigen and mRNA. Leishmania donovani proteins were used in the development of an ELISA for identifying human screening donors. A panel of human blood donors with a history of visceral leishmaniasis was obtained from Sudanese donors. The serum antibody reactivity of the donors was tested by ELISA and serum donors showing high antibody reactivity against Leishmania donovani amastigote proteins were selected for use in screening of a Leishmania donovani amastigote cDNA library. Two cDNA libraries were constructed in parallel from both promastigote and amastigote mRNA. The amastigote cDNA library was screened using the panel of ELISA selected human blood donors. of 128 isolated cDNA-clones, 6 clones were selected for sequencing and 4 of the 6 clones were selected for further investigations based on the sequence data. Of the 4 clones, 3 were shown to be full length clones as judged from Northern blots. The coding DNA sequence corresponded to 4 different proteins; a) a 58.1 kDa protein related to the kinesin-like protein superfamily, b) a 47.7 kDa ribosomal protein L3, c) a 27.9 kDa protein with no apparent homology to known protein sequences, and d) a 27.2 kDa protein, a 20S proteasome subunit, member of the multi catalytic proteinase complex. Fusion proteins were induced from the 4 clones and fusion protein was purified using a modified standard protocol. All 4 fusion proteins reacted in a Western blot with the serum pool originally used in the cDNA library screening process, indicating their immunological integrity    Involved research institution(s) Centre for Medical Parasitology, Institute of Medical Microbiology and Immunology, University of Copenhagen Department of Infectious Diseases, University of Copenhagen   Supervisor(s) Thor Theander, Centre for Medical Parasitology, Copenhagen   Correspondence