Studies on the development of protective immunity with the 150-kDa lectin of Entamoeba histolytica

我们最近鉴定了一个150 kda的溶组织内阿米巴表面抗原，作为半乳糖和n -乙酰- d -半乳糖抑制凝集素的中间亚基（Igl）。本研究在大肠杆菌中制备了除信号序列[氨基酸（aa） 14- 1088]外的全长Igl和3个Igl片段，即n端部分（aa 14-382）、中间部分（aa 294-753）和c端部分（aa 603- 1088），并采用酶联免疫吸附试验（ELISA）检测了这些重组蛋白与阿米巴病患者血清的反应性。有症状的阿米巴肝脓肿或阿米巴结肠炎患者的血清、无症状的囊肿过境者的血清、其他原生动物感染个体的血清以及健康对照者的血清被使用。ELISA检测重组全长Igl的灵敏度为90%，特异性为94%。用3个片段作为抗原进行ELISA检测时，n端敏感性为56%，中间敏感性为92%，c端敏感性为97%。三种抗原在n端特异性为96%，在中间和c端特异性均为99%。用F-Igl、M-Igl和C-Igl作为抗原，无症状囊肿传者血清均无阴性。另一方面，在使用M-Igl和C-Igl的ELISA中，仅在结肠炎病例中检测到假阴性结果。这些观察结果表明，识别M-Igl和C-Jgl表位的抗体可能具有阻止滋养体侵入宿主组织的功能，并且Igl的羧基末端是一种特别有用的抗原，可用于阿米巴病的血清诊断。当检测粘附抑制单克隆抗体对各种重组片段的反应性时，其中一个表位定位在aa 989- 10,088的部分。天然Igl免疫能显著抑制仓鼠肝脓肿的形成，而F-Igl免疫则不能，提示Igl糖链上的表位也可能对诱导保护性免疫起重要作用。从异棘内阿米巴中克隆到编码1,110和1,106 aa的两个Igl基因，并进行了进一步分析。少

We have recently identified a 150-kDa surface antigen of Entamoeba histolytica as an intermediate subunit (Igl) of galactose-and N-acetyl-D-galactosamine-inhibitable lectin. In the present study, full-length Igl except for the signal sequences [amino acid (aa) 14-1,088] and three fragments of Igl, the N-terminal part (aa 14-382), middle part (aa 294-753), and C-terminal part (aa 603-1,088), were prepared in Escherichia coli and the reactivity of these recombinant proteins with sera from patients with amebiasis was examined by means of enzyme-linked immunosorbent assay (ELISA). Sera from symptomatic patients with amebic liver abscess or amebic colitis, sera from asymptomatic cyst passers, sera from individuals with other protozoan infections, and sera from healthy controls were used. Sensitivity and specificity of the recombinant full-length Igl in the ELISA were 90% and 94% respectively. When three fragments were used as antigens in the ELISA, sensitivities were 56% in the N-terminus, … More 92% in the middle part, and 97% in the C-terminus. Specificities of the three antigens were 96% in the N-terminus and 99% in both the middle and C-terminus fragments. None of the sera from asymptomatic cyst passers was negative when F-Igl, M-Igl and C-Igl were used as the antigen. On the other hand, in the cases of ELISA using M-Igl and C-Igl, false-negative results were detected in only the colitis cases. These observations suggest that the antibodies which recognized the epitopes located in M-Igl and C-Jgl may function to prevent the invasion of trophozoites into host tissues and that the carboxyl terminus of Igl is an especially useful antigen for the serodiagnosis of amebiasis. When the reactivity of adherence-inhibitory monoclonal antibodies to various recombinant fragments was also examined, one of the epitope was localized in the part of aa 989-1, 088. Liver abscess formation of hamsters was significantly inhibited by the immunization with native Igl, but not with the F-Igl, suggesting that the epitope (s) on sugar chain(s) of Igi may also be important for the induction of protective immunity. Two Igl genes, coding 1, 110 and 1, 106 aa, were also cloned from Entamoeba dispar and were further analyzed. Less