| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1988: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1987: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
The cDNA encoding antigenic protein of Toxoplasma gondii was cloned to utilize as a diagnostic antigen. Total RNA extracted from T. gondii tachyzoite was in vitro translated using rabbit reticulocyte lysate for the identification of antigenic protein. Translation products were immunoprecipitated with T. gondii-infected human serum. Three major antigenic proteins with molecular weights of 27,000, 31,000 and 35,000 were detected by the SDS-PAGE autoradiography. The predominant antigenic polyprptide among them has a molecular weight of 27,000. cDNA library was constructed in gtll expression vector. cDNA clones encoding 27 kDa antigenic polypeptide were immunoscreened using infected human serum. Twenty six positive clones were isolated from 4x10^4. Monospecific antibody to respective clone was epitope-selected from polyclonal antiserum and reacted with in vitro translation products to identify cDNA clone encoding 27 kDa-antigenic polypeptide. The molecular weight of antigenic protein was determined by the SDS-PAGE autoradiography. As a result, Tg18 was identified as a cDNA clone encoding 2m kDa-antigenic polypeptide. Southern blot assay demonstrated that the gene encoding 27 kDa antigen was located in approximately 13 kb EcoRI fragment of genomic DNA. Northern blot assay revealed that the mRNA for 27 kDa antigen was 1400 nucleotides in size. Native antigens with molecular weights of 32,000 and 27,000 were detected by the Western blot assay. Furthermore, gtll -galactosidase fusion protein synthesized in E. coli had no cross-reactivity with the other parasite-infected serum. Tg18 cDNA is 739 bp in length containing open reading frame encoding 174 amino acids. Genbank nucleic acid and NBRF data banks reveal no sequence homology.
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