2001 Fiscal Year Final Research Report Summary
Production of recombinant human monoclonal antibodies ot a surface lection Entamoeba histolytica
| Project/Area Number |
12670242
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
寄生虫学(含医用動物学)
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| Research Institution | Tokai University |
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Principal Investigator |
TACHIBANA Hiroshi Tokai University, School fo Medicine, Associate Professor, 医学部, 助教授 (10147168)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Entamoeba histolytica / human monoclonal antibody / lection / asymptomatic cyst passer / immunoglobulin gene |
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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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Research Products
(18 results)
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[Publications] Tachibana, H., Cheng, X.-J., Kobayashai, S., Fujita, Y. and Udono, T.: "Entamoeba dispar, but not E. histolytica, detected in a colony of chimpanzees in Japan."Parasitol. Res.. 86(7). 537-541 (2000)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Tachibana, H., Cheng, X.-J., Kobayashai, S., Matsubayashi, N., Gotoh, S. and Matsubayashi, K.: "High prevalence of infection with Entamoeba dispar, but not E. histolytica, in captive macaques."Parasitol. Res.. 87(1). 14-17 (2001)
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「研究成果報告書概要(欧文)」より
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[Publications] Cheng, X. -J., Hughes, M. A., Huston, C. D., Loftus, B., Gilchrist, C. A., Lockhart, L. A., Ghoshi, S., Miller-Sims, V., Mann, B. J.j Petri, W. A., Jr. and Tachibana, H.: "Intermediate subunit of the Gal/GalNAc lectin of Entamoeba histolytica is a member of a gene family containing multiple CXXC sequence motifs."Infect. Immun.. 69(9). 5892-5898 (2001)
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「研究成果報告書概要(欧文)」より
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[Publications] Tanyksel, M., Tachinaba, H., and Petri, W. A., Jr.: "Amebiasis, and Emerging Disease."Emerging Infections 5, Scheld, W. M., Craig, W. A. and Hughes, J. M. eds., ASM Press, Washington, D.C.. 197-212 (2001)
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「研究成果報告書概要(欧文)」より
