Cloning and expression of mitochondrial heat shock protein 70 of Trypanosoma congolense and potential use as a diagnostic antigen
Cloning and expression of mitochondrial heat shock protein 70 of Trypanosoma congolense and potential use as a diagnostic antigen
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DOI：
10.1128/cdli.10.5.926-933.2003
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发表时间：
2003-09-01
期刊：
影响因子：
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通讯作者：
Igarashi, I
中科院分区：
文献类型：
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作者：
Bannai, H;Sakurai, T;Igarashi, I
检测了刚果锥虫线粒体热休克蛋白70 （MTP）作为诊断抗原的能力。用单克隆抗体（MAb） 10F9对刚果螺旋体原环型（PCF） cDNA文库进行免疫筛选，获得1个cDNA克隆。cDNA克隆包含一个1977bp的开放阅读框，编码659个氨基酸组成的多肽。Southern印迹分析表明，在单倍体基因组中至少有3个MTP基因拷贝。干扰MTP RNA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在血流形态（BSF）和PCF中均有表达。MAb 10F9识别的b细胞表位位于C端206个氨基酸内。根据蛋白质提取的条件，MTP被内源性蛋白酶裂解成更小的多肽。然而，MTP的c端表位即使在切割后也保持了高度的抗原性。用截断的重组MTP酶联免疫吸附法检测抗体，发现在实验感染小鼠血清中存在抗MTP抗体。因此，MTP可能是一种有用的抗原，用于原发性刚果弓形虫感染的血清诊断。
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.