Tachibana H, Cheng X J, Watanabe K, Takekoshi M, Maeda F, Aotsuka S, Kaneda Y, Takeuchi T, Ihara S
Departments of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan.
Clin Diagn Lab Immunol. 1999 May;6(3):383-7. doi: 10.1128/CDLI.6.3.383-387.1999.
Genes coding for human antibody Fab fragments specific for Entamoeba histolytica were cloned and expressed in Escherichia coli. Lymphocytes were separated from the peripheral blood of a patient with an amebic liver abscess. Poly(A)+ RNA was isolated from the lymphocytes, and then genes coding for the light chain and Fd region of the heavy chain were amplified by a reverse transcriptase PCR. The amplified DNA fragments were ligated with a plasmid vector and were introduced into Escherichia coli. Three thousand colonies were screened for the production of antibodies to E. histolytica HM-1:IMSS by an indirect fluorescence-antibody (IFA) test. Lysates from five Escherichia coli clones were positive. Analysis of the DNA sequences of the five clones showed that three of the five heavy-chain sequences and four of the five light-chain sequences differed from each other. When the reactivities of the Escherichia coli lysates to nine reference strains of E. histolytica were examined by the IFA test, three Fab fragments with different DNA sequences were found to react with all nine strains and another Fab fragment was found to react with seven strains. None of the four human monoclonal antibody Fab fragments reacted with Entamoeba dispar reference strains or with other enteric protozoan parasites. These results indicate that the bacterial expression system reported here is effective for the production of human monoclonal antibodies specific for E. histolytica. The recombinant human monoclonal antibody Fab fragments may be applicable for distinguishing E. histolytica from E. dispar and for use in the serodiagnosis of amebiasis.
编码对溶组织内阿米巴具有特异性的人抗体Fab片段的基因被克隆并在大肠杆菌中表达。从一名阿米巴肝脓肿患者的外周血中分离淋巴细胞。从淋巴细胞中分离出聚腺苷酸（Poly(A)+）RNA，然后通过逆转录聚合酶链反应（RT-PCR）扩增编码轻链和重链Fd区的基因。将扩增的DNA片段与质粒载体连接，并导入大肠杆菌。通过间接荧光抗体（IFA）试验筛选3000个菌落，以检测其是否产生针对溶组织内阿米巴HM-1:IMSS的抗体。五个大肠杆菌克隆的裂解物呈阳性。对五个克隆的DNA序列分析表明，五个重链序列中的三个和五个轻链序列中的四个彼此不同。当通过IFA试验检测大肠杆菌裂解物对九种溶组织内阿米巴参考菌株的反应性时，发现三个具有不同DNA序列的Fab片段与所有九种菌株反应，另一个Fab片段与七种菌株反应。四种人单克隆抗体Fab片段均未与迪斯帕内阿米巴参考菌株或其他肠道原生动物寄生虫发生反应。这些结果表明，本文报道的细菌表达系统对于生产针对溶组织内阿米巴的人单克隆抗体是有效的。重组人单克隆抗体Fab片段可用于区分溶组织内阿米巴和迪斯帕内阿米巴，并用于阿米巴病的血清学诊断。
