| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
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.
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