Production of recombinant human monoclonal antibodies ot a surface lection Entamoeba histolytica

溶组织内阿米巴可引起阿米巴结肠炎和/或肠外脓肿。然而，无症状病例仅通过E。粪便中存在溶组织性包囊。在这样的包囊传播者中，阻止滋养体侵入组织的保护性抗体可能是可行的。因此，我们从一名无症状的囊肿者的外周血淋巴细胞中构建了一个免疫球蛋白基因文库，并在大肠杆菌中制备了特异性的人免疫球蛋白单抗。细菌菌落在硝酸纤维素膜上印迹，筛选产生抗溶组织埃希氏菌抗体Fab片段的克隆。通过首先加入滋养体的裂解物，然后从有症状的患者那里获得标记的多克隆抗体来检测阳性克隆。对其中一个阳性克隆CP33进行了分析。斑点印迹分析表明，CP33可识别260 kDa Gal/GalNAc凝集素的半胱氨酸富集区。用CP33预处理溶组织圆线虫滋养体，再与人红细胞孵育，可显著抑制红细胞吞噬功能。用CP33的重链基因对文库中的轻链基因进行重组，得到了多个由不同轻链基因组成的Fab克隆。其中有3个克隆对溶组织埃希氏菌的红细胞吞噬功能有明显的抑制作用，与CP33相似。肠道中的这些重组人类抗体和/或产生抗体的细菌可能被证明对阿米巴病的预防和治疗有效。为了将这些Fab片段应用于诊断，设计并构建了Fab与碱性磷酸酶(PhoA)融合蛋白在大肠杆菌中的表达载体。将PhoA基因融合到编码重链Fd区的基因的3‘端。以CP-33的kappa和fd基因作为人源抗体基因。人源性单抗-PhoA结合物在细菌中的表达是可能的。

Entamoeba histolytica can cause amebic colitis and/or extraintestinal abscesses. However, asymptomatic cases only passing E.. Histolytica cysts in feces exist. In such cyst passers, protective antibodies which block invasion of trophozoites into tissue may be feasible. We have therefore constructed an immunoglobulin gene library from peripheral lymphocytes of an asymptomatic cyst passer and prepared E. histolytica-specific human monoclonal antibodies in Escherichia coll. Bacterial colonies were blotted on nitrocellulose membranes and screened for clones producing antibody Fab fragments to E. histolytica. Positive clones were detected by first adding lysates of trophozoites followed by labeled polyclonal antibodies obtained from a symptomatic patient. One of the positive clones, CP33, was analyzed. Dot blot analysis demonstrated that CP33 recognized cysteine-rich domain of a 260-kDa Gal/GalNAc lectin. When E. histolytica trophozoites were pretreated with CP33 and then incubated with human erythrocytes, erythrophagocytosis was significantly inhibited. Reshuffling the light chain genes of the library with the heavy chain gene of CP33, many Fab clones consisting of different light chain genes were obtained. Of these, 3 clones showed significant inhibitory effects on erythrophagocytosis of E. histolytica, comparable to CP33. These recombinant human antibodies and/or antibody-producing bacteria in the gut may prove effective for the prevention and treatment of amebiasis. For application of these Fab fragments for diagnostic purposes, an expression vector to produce a fusion protein of Fab and alkaline phosphatase (PhoA) in E. coli was designed and constructed. E. coli PhoA gene was fused to the 3' terminus of the gene coding the heavy chain Fd region. As human antibody genes, the Kappa and Fd genes from CP-33, were used. The bacterial expression of a human monoclonal antibody-PhoA conjugate specific for E. histolytica was possible.