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性结肠炎和/或肠外脓肿。然而，无症状病例仅通过E.粪便中存在溶组织囊肿。在这种包囊传递者中，阻断滋养体侵入组织的保护性抗体可能是可行的。因此，我们构建了一个免疫球蛋白基因文库，从外周淋巴细胞的无症状囊肿通过和制备E。将细菌菌落在硝酸纤维素膜上印迹，筛选产生抗大肠杆菌抗体Fab片段的克隆。溶组织剂通过首先加入滋养体的裂解物，然后加入从有症状的患者获得的标记的多克隆抗体来检测阳性克隆。对其中一个阳性克隆CP 33进行了分析。斑点杂交分析表明，CP 33识别的260 kDa的Gal/GalNAc凝集素的富含半胱氨酸的结构域。当E.用CP 33预处理溶组织滋养体，然后与人红细胞孵育，红细胞吞噬作用显著抑制。将文库中的轻链基因与CP 33的重链基因进行重组，获得了多个由不同轻链基因组成的Fab克隆。其中3个克隆对E.溶组织菌，与CP 33相当。这些重组人抗体和/或肠道中产生抗体的细菌可能被证明对预防和治疗阿米巴病有效。为了将这些Fab片段用于诊断目的，在大肠杆菌中构建了产生Fab和碱性磷酸酶（PhoA）融合蛋白的表达载体。coli中进行构建。E. coli PhoA基因与编码重链Fd区的基因的3'末端融合。作为人抗体基因，使用来自CP-33的Kappa和Fd基因。人源性抗大肠杆菌单克隆抗体-PhoA偶联物在大肠杆菌中的表达组织溶解是可能的。

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.