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Clinical and Diagnostic Laboratory Immunology, September 2003, p. 926-933, Vol. 10, No. 5
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.5.926-933.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cloning and Expression of Mitochondrial Heat Shock Protein 70 of Trypanosoma congolense and Potential Use as a Diagnostic Antigen

Hiroshi Bannai, Tatsuya Sakurai, Noboru Inoue,* Chihiro Sugimoto, and Ikuo Igarashi

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan

Received 27 December 2002/ Returned for modification 17 April 2003/ Accepted 12 June 2003

The ability to use mitochondrial heat shock protein 70 (MTP) of Trypanosoma congolense as a diagnostic antigen was examined. One cDNA clone was obtained by immunoscreening of a T. congolense procyclic form (PCF) cDNA library with monoclonal antibody (MAb) 10F9. The cDNA clone contained an open reading frame of 1,977 bp encoding a polypeptide consisting of 659 amino acids. Southern blotting analysis indicated that there were at least three copies of the MTP gene in the haploid genome. Interference of the MTP RNA resulted in complete inhibition, which indicated that MTP is essential at the PCF stage. Northern and Western blotting analyses revealed that MTP is expressed both in the bloodstream form (BSF) and in PCF. The B-cell epitope recognized by MAb 10F9 was located within 206 amino acids from the C terminus. Depending on the conditions of protein extraction, MTP was cleaved into smaller polypeptides by endogenous proteases. However, the C-terminal epitope of MTP was preserved with a high degree of antigenicity, even after cleavage. Antibody detection by enzyme-linked immunosorbent assay with the truncated recombinant MTP revealed that anti-MTP antibodies exist in experimentally infected mouse sera. Thus, MTP may be useful as an antigen for the serodiagnosis of primary T. congolense infection.

* Corresponding author: Mailing address: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan. Phone: 81-155-49-5647. Fax: 81-155-49-5643. E-mail: ircpmi{at}obihiro.ac.jp.

Clinical and Diagnostic Laboratory Immunology, September 2003, p. 926-933, Vol. 10, No. 5
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.5.926-933.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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