Bannai Hiroshi, Sakurai Tatsuya, Inoue Noboru, Sugimoto Chihiro, Igarashi Ikuo
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan.
Clin Diagn Lab Immunol. 2003 Sep;10(5):926-33. doi: 10.1128/cdli.10.5.926-933.2003.
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.
对刚果锥虫线粒体热休克蛋白70（MTP）作为诊断抗原的能力进行了检测。通过用单克隆抗体（MAb）10F9对刚果锥虫前循环型（PCF）cDNA文库进行免疫筛选，获得了一个cDNA克隆。该cDNA克隆包含一个1977 bp的开放阅读框，编码一个由659个氨基酸组成的多肽。Southern印迹分析表明，单倍体基因组中至少有三个MTP基因拷贝。MTP RNA的干扰导致完全抑制，这表明MTP在PCF阶段是必不可少的。Northern和Western印迹分析显示，MTP在血流型（BSF）和PCF中均有表达。MAb 10F9识别的B细胞表位位于距C末端206个氨基酸范围内。根据蛋白质提取条件，MTP被内源性蛋白酶切割成较小的多肽。然而，即使在切割后，MTP的C末端表位仍保留高度抗原性。用截短的重组MTP通过酶联免疫吸附测定法检测抗体，结果显示在实验感染小鼠血清中存在抗MTP抗体。因此，MTP可能作为原发性刚果锥虫感染血清诊断的抗原。
