Hostname: page-component-78c5997874-lj6df Total loading time: 0 Render date: 2024-11-15T11:17:54.227Z Has data issue: false hasContentIssue false
  • English
  • Français

Construction and characterization of recombinant single-chain variable fragment antibodies against Toxoplasma gondii MIC2 protein

Published online by Cambridge University Press:  27 July 2005

L.-N. HOE ,
K.-L. WAN  and
S. NATHAN
L.-N. HOE
Affiliation:
Centre for Gene Analysis and Technology, School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor D. E., Malaysia
K.-L. WAN
Affiliation:
Centre for Gene Analysis and Technology, School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor D. E., Malaysia
S. NATHAN
Affiliation:
Centre for Gene Analysis and Technology, School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor D. E., Malaysia
Get access Rights & Permissions [Opens in a new window]

Abstract

The protozoan parasite Toxoplasma gondii produces a family of microneme proteins that are thought to play diverse roles in aiding the parasite's intracellular existence. Among these, TgMIC2 has a putative function in parasite adhesion to the host cell to initiate the invasion process. The invasion process may be localized and inhibited by monoclonal antibodies against the protein(s) involved. Here we report on the construction of a phage-displayed single-chain variable fragment (scFv) library from mice immunized with whole T. gondii parasites. The library was subsequently panned against recombinant TgMIC2 (rpTgMIC2) and 2 different groups of antibody clones were obtained, based on fingerprinting and sequencing data. The expressed recombinant scFv antibody was able to recognize rpTgMIC2 in a Western blot detection experiment. These results show that the phage display technology allows quick and effective production of monoclonal antibodies against parasite antigens. By panning the scFv-displayed library, we should be able to obtain a plethora of multi-functional scFv antibodies towards T. gondii proteins.

Keywords

phage displaymonoclonal antibodiesrecombinant proteintoxoplasmosis
Type
Research Article
Information
Parasitology , Volume 131 , Issue 6 , December 2005 , pp. 759 - 768
Copyright
© 2005 Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Andris-Widhopf, J., Steinberger, P., Fuller, R., Rader, C. and Barbas, C. F. ( 2001). Generation of antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences. In Phage Display: A Laboratory Manual ( ed. Barbas, C. F., Burton, D. R., Scott, J. K. and Silverman, G. J.), pp. 9.1–9.113. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Arap, W., Pasqualini, R. and Ruoslahti, E. ( 1998). Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279, 377–380.CrossRefGoogle Scholar
Barbas, C. F., Kang, A., Lerner, R. and Benkovic, S. ( 1991). Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proceedings of the National Academy of Sciences, USA 88, 7978–7982.CrossRefGoogle Scholar
Beghetto, E., Nielsen, H. V., Del Porto, P., Buffolano, W., Guglietta, S., Felici, F., Petersen, E. and Gargano, N. ( 2005). A combination of antigenic regions of Toxoplasma gondii microneme proteins induces protective immunity against oral infection with parasite cysts. Journal of Infectious Diseases 191, 637–645.CrossRefGoogle Scholar
Bera, T. K., Kennedy, P. E., Berger, E. A., Barbas, C. F. III and Pastan, I. ( 1998). Specific killing of HIV-infected lymphocytes by a recombinant immunotoxin directed against the HIV-1 envelope glycoprotein. Molecular Medicine 4, 384–391.Google Scholar
Burton, D. R. ( 2001). Antibody libraries. In Phage Display: A Laboratory Manual ( ed. Barbas, C. F., Burton, D. R., Scott, J. K. & Silverman, G. J.), pp. 3.1–3.18. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Carruthers, V. B. ( 2002) Host cell invasion by the opportunistic pathogen Toxoplasma gondii. Acta Tropica 81, 111–122.CrossRefGoogle Scholar
Carruthers, V. B. and Sibley, L. D. ( 1997). Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. European Journal of Cellular Biology 73, 114–123.Google Scholar
Carzaniga, R., Fiocco, D., Bowyer, P. and O'conell, R. J. ( 2002). Localization of melanin in conidia of Alternaria alternata using phage display antibodies. Molecular Plant and Microbe Interaction, 15, 216–224.CrossRefGoogle Scholar
Chowdhury, P. S. and Pastan, I. ( 1999). Improving antibody affinity by mimicking somatic hypermutation in vitro. Nature Biotechnology 17, 568–572.CrossRefGoogle Scholar
Donovan, R. S., Robinson, C. W. and Glick, B. R. ( 2000). Optimizing the expression of a monoclonal antibody fragment under the transcriptional control of the Escherichia coli lac promoter. Canadian Journal of Microbiology 46, 532–541.CrossRefGoogle Scholar
Dubey, J. P. and Beattie, C. P. ( 1988). Toxoplasmosis of Animals and Man. CRC Press, Boca Raton, Fl, USA.
Elia, M., Andris-Widhopf, J., Fuller, R. and Barbas, C. F. ( 2001). Production and purification of Fab and scFv. In Phage Display: A Laboratory Manual ( ed. Barbas, C. F., Burton, D. R., Scott, J. K. & Silverman, G. J.), pp. 12.1–12.26. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Elsaid, M. M. A., Vitor, R. W. A., Frezard, F. J. G. and Martins, M. S. ( 1999). Protection against toxoplasmosis in mice immunized with antigens of Toxoplasma gondii incorporated into liposomes. Memorias do Instituto Oswaldo Cruz, Rio de Janeiro 94, 485–490.CrossRefGoogle Scholar
Harlow, E. and Lane, D. ( 1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Harper, J. M., Hoff, E. F. and Carruthers, V. B. ( 2004). Multimerization of the Toxoplasma gondii MIC2 integrin-like A-domain is required for binding to heparin and human cells. Molecular and Biochemical Parasitology 134, 201–212.CrossRefGoogle Scholar
Hawkins, R. E., Russell, S. J., Baier, M. and Winter, G. ( 1993). The contribution of contact and non-contact residues of antibody in the affinity of binding to antigen. The interaction of mutant D1.3 antibodies with lysozyme. Journal of Molecular Biology 234, 958–964.CrossRefGoogle Scholar
Huston, J. S., Levinson, D., Mudgett-hunter, M., Tai, M.-S., Novotny, J., Margolies, M. J., Ridge, R. J., Bruccoleri, R. E., Haber, E., Crea, R. and Oppermann, H. ( 1988). Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proceedings of the National Academy of Sciences, USA 85, 5879–5883.CrossRefGoogle Scholar
Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S. and Foeller, C. ( 1991). Sequences of Proteins of Immunological Interest. 5th Edn. National Institutes of Health, Maryland.
Laemmli, U. K. ( 1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, London 227, 680.CrossRefGoogle Scholar
Le Gall, F., Kipriyanov, S. M., Moldenhauer, G. and Little, M. ( 1999). Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding. FEBS Letters 453, 164–168.CrossRefGoogle Scholar
Low, N. M., Holliger, P. and Winter, G. ( 1996). Mimicking somatic hypermutation: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain. Journal of Molecular Biology 260, 359–368.CrossRefGoogle Scholar
Oh, M. S., Kim, K. S., Jang, Y. K., Maeng, C. Y., Min, S. H., Jang, M. H., Yoon, S. O., Kim, J. H. and Hong, H. J. ( 2003). A new epitope tag from Hepatitis B virus preS1 for immunodetection, localization and affinity purification of recombinant proteins. Journal of Immunological Methods 283, 77–89.CrossRefGoogle Scholar
Prigione, I., Facchetti, P., Lecordier, L., Deslee, D., Chiesa, S., Cesbron-Delauw, M. F. and Pistoia, V. ( 2000). T cell clones raised from chronically infected healthy humans by stimulation with Toxoplasma gondii excretory-secretory antigens cross-react with live tachyzoites: characterization of the fine antigenic specificity of the clones and implications for vaccine development. Journal of Immunology 164, 3741–3748.CrossRefGoogle Scholar
Robben, J., Hertveldt, K., Bosmans, E. and Volckaert, G. ( 2002). Selection and identification of dense granule antigen GRA3 by Toxoplasma gondii whole genome phage display. Journal of Biological Chemistry 277, 17544–17547.CrossRefGoogle Scholar
Robson, K. J. H., Hall, J. R. S., Jennings, M. W., Harris, T. J. R., Marsh, K., Newbold, C. I., Tate, V. E. and Weatherall, D. J. ( 1988). A highly conserved amino acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stages of a human malaria parasite. Nature, London 335, 79–82.CrossRefGoogle Scholar
ROCHE MOLECULAR CHEMICALS ( 1999). Anti-[Ha]-Peroxidase.
Shabat, D., Lode, H. N., Pertl, U., Reisfeld, R. A., Rader, C. R., Lerner, R. A. and Barbas, C. F. III. ( 2001). In vivo activity in a catalytic antibody-prodrug system: antibody catalyzed etoposide prodrug activation for selective chemotherapy. Proceedings of the National Academy of Sciences, USA 98, 7528–7533.CrossRefGoogle Scholar
Smith, G. P. ( 1985). Filamentous fusion phage: novel expression vectors that display cloned antigens on the surface of the virion. Science 288, 1315–1317.CrossRefGoogle Scholar
Studier, F. W., Rosenberg, A. H., Dunn, J. J. and Dubendorff, J. W. ( 1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology 185, 60–89.CrossRefGoogle Scholar
Tomley, F. M., Clarke, L. E., Kawazoe, U., Dijkema, R. and Kok, J. J. ( 1991). Sequence of the gene encoding an immunodominant microneme protein of Eimeria tenella. Molecular and Biochemical Parasitology 49, 277–288.CrossRefGoogle Scholar
Towbin, H., Staehelin, T. and Gordon, J. ( 1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350.CrossRefGoogle Scholar
Trottein, F., Triglia, T. and Cowman, A. F. ( 1995). Molecular cloning of a gene from Plasmodium falciparum that codes for a protein sharing motif found in adhesive molecules from mammals and Plasmodia. Molecular and Biochemical Parasitology 74, 129–141.CrossRefGoogle Scholar
Wan, K.-L., Blackwell, J. M. and Ajioka, J. W. ( 1996). Toxoplasma gondii expressed sequence tags: insights into tachyzoite gene expression. Molecular and Biochemical Parasitology 75, 179–186.CrossRefGoogle Scholar
Wan, K.-L., Carruthers, V. B., Sibley, L. D. and Ajioka, J. W. ( 1997). Molecular characterization of an expressed sequence tag locus of Toxoplasma gondii encoding the micronemal protein MIC2. Molecular and Biochemical Parasitology 84, 203–214.CrossRefGoogle Scholar
White, G. P., Meeusen, E. N. and Newton, S. E. ( 2001). A single-chain variable region immunoglobulin library from the abomasal lymph node of sheep infected with the gastrointestinal nematode parasite Haemonchus contortus. Veterinary Immunology and Immunopathology, 78, 117–129.CrossRefGoogle Scholar