Molecular cloning, expression, and serological evaluation of an, 8-kilodalton subunit of antigen B from Echinococcus multilocularis
Molecular cloning, expression, and serological evaluation of an, 8-kilodalton subunit of antigen B from Echinococcus multilocularis
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DOI：
10.1128/jcm.42.3.1082-1088.2004
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发表时间：
2004-03-01
影响因子：
9.4
通讯作者：
Ito, A
中科院分区：
文献类型：
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作者：
Mamuti, W;Yamasaki, H;Ito, A
从大肠杆菌中分离得到编码泡球蚴抗原B 8-kDa亚基的全长cDNA和基因组DNA（命名为EmAgB 8/1）。多室后绦虫cDNA文库和原头节基因组DNA文库。cDNA克隆的开放阅读框编码包含85个氨基酸和20个氨基酸的NH 2-末端信号序列的多肽，这在天然抗原的N-末端测序后得到证实。逆转录-PCR分析表明，EmAgB 8/1基因主要在E.多房的该基因由两个外显子（编码信号序列和成熟蛋白）组成，由一个91 bp的内含子分隔。成熟的形式在大肠杆菌中表达，其抗原反应性进行了比较与对应物，一个8-kDa的抗原B亚单位从细粒棘球蚴（EgAgB 8/1）通过蛋白质印迹和酶联免疫吸附试验（ELISA）与患者的血清样品确认有囊型包虫病（CE）和泡型包虫病（AE）。重组EmAgB 8/1在CE患者和AE患者血清中的阳性率分别为81.3%（65/80）和40.6%（26/64），而重组EgAgB 8/1在CE和AE患者血清中的阳性率分别为86%（43/50）和42%（19/45）。EmAgB 8/1和EgAgB 8/1的ELISA阳性率相似，分别为88%（44/50）和37.8%（17/45）。统计学分析表明，EmAgB 8/1与EgAgB 8/1对棘球蚴病的血清学诊断敏感性相当。与囊虫病患者血清无交叉反应，而囊虫病患者血清在用天然抗原进行血清学诊断时常发生交叉反应。
Full-length cDNA and genomic DNA encoding an 8-kDa subunit of antigen B from Echinococcus multilocularis (designated EmAgB8/1) were isolated from an E. multilocularis metacestode cDNA library and a protoscolex genomic DNA library, respectively. The open reading frame of the cDNA clone encodes a polypeptide comprising 85 amino acids with a 20-amino-acid NH2-terminal signal sequence, which was confirmed following N-terminal sequencing of the native antigen. Reverse transcription-PCR analysis revealed that the clone encoding EmAgB8/1 is predominantly transcribed in larval E. multilocularis. The gene consists of two exons (encoding the signal sequence and mature protein) separated by a 91-bp intron. The mature form was expressed in Escherichia coli, and its antigenic reactivity was compared with that of a counterpart, an 8-kDa subunit of antigen B from Echinococcus granulosus (EgAgB8/1) by Western blotting and enzyme-linked immunosorbent assay (ELISA) with serum samples from patients confirmed to have cystic echinococcosis (CE) and alveolar echinococcosis (AE). Recombinant EmAgB8/1 showed positive reactions in Western blots with 81.3% (65 of 80) of serum samples from CE patients and 40.6% (26 of 64) of serum samples from AE patients, while recombinant EgAgB8/1 showed positive reactions with 86% (43 of 50) and 42% (19 of 45) of the serum samples from these CE and AE patients, respectively. By the ELISA, both EmAgB8/1 and EgAgB8/1 exhibited similar positive reactions with 88% (44 of 50) of serum samples from CE patients and 37.8% (17 of 45) serum samples from AE patients. Statistical analysis revealed that the sensitivity of EmAgB8/1 was comparable to that of EgAgB8/1 for the serodiagnosis of echinococcal diseases. There was no cross-reaction with sera from patients with cysticercosis, which often cross-react when native antigens are used for serodiagnosis.