DOI QR코드

DOI QR Code

Cloning and Expression of Partial Japanese Flounder (Paralichthys olivaceus) IgD

  • Choi, Dae-Han (Department of Aquatic Medicine, Kunsan National University) ;
  • Jang, Han-Na (Department of Aquatic Medicine, Kunsan National University) ;
  • Ha, Dae-Mang (Department of Aquatic Medicine, Kunsan National University) ;
  • Kim, Jae-Wha (Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology) ;
  • Oh, Chan-Ho (Department of Food Biotechnology, Woosuk University) ;
  • Choi, Sang-Hoon (Department of Aquatic Medicine, Kunsan National University)
  • Published : 2007.07.31

Abstract

The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7$\cdot}$Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.

Keywords

cDNA;ELISA;Flounder;IgD;Recombinant protein

References

  1. Aoki, T., Hirono, I., Kim, M.-G., Katagiri, T., Tkuda, Y. and Toyohara, H. (2000) Identification of viral induced genes in Ig+ leucocytes of Japanese flounder Paralichthys olivaceus, by differential hybridization with subtracted and un-subtracted cDNA probes. Fish Shellfish Immunol. 10, 623-630. https://doi.org/10.1006/fsim.2000.0279
  2. Aoki, T., Nam, B. H. and Hirono, I. (1999) Sequence of 596 cDNA clones (565,977 bp) of Japanese flounder Paralichthys olivaceus leukocytes infected with hirame rhabdovirus, Mar. Biotechnol. 1, 477-488. https://doi.org/10.1007/PL00011804
  3. Bengten, E., Quiniou, M., Stuge, T. B., Katagiri, T., Miller, N. W., Clem, L. W., Warr, G. W. and Wilson, M. (2002) The IgH locus of the channel catfish, Ictalurus punctatus, contains multiple constant region gene sequences: different genes encode heavy chains of membrane and secreted IgD. J. Immunol. 169, 2488-2497. https://doi.org/10.4049/jimmunol.169.5.2488
  4. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  5. Choi, S. H., Park, K. H. and Yoon, J. M. (2002) Characterization of Carp (Cyprinus carpio L.) immunoglobulin structure. Asian-Aust. J. Anim. Sci. 15, 290-296. https://doi.org/10.5713/ajas.2002.290
  6. DeLuca, D. Wilson, M. and Warr, G. W. (1983) Lymphocyte heterogeneity in the trout, Salmo gairdneri, defined with monoclonal antibodies to IgM. Eur. J. Immunol. 13, 546-551. https://doi.org/10.1002/eji.1830130706
  7. Estevez, J., Leiro, J. M., Santamarina, T., Dominquez, J. and Ubeira, F. M. (1994) Monoclonal antibodies to turbot (Scophthalmus maximus) immunoglobulins: characterization and applicability in immunoassays. Vet. Immunol. Immunopathol. 41, 353-366. https://doi.org/10.1016/0165-2427(94)90107-4
  8. Graham, S. C. and Secombes, J. (1990) Cellular requirements for lymphokine secretion by rainbow trout Salmo gairdneri leucocytes. Dev. Comp. Immunol. 30, 59-68.
  9. Hirono, I., Nam, B. H., Enomoto, J., Uchino, K. and Aoki, T. (2003) Cloning and characterization of a cDNA encoding Japanese flounder Paralichthys olivaceus IgD, Fish Shellfish Immunol. 15, 63-70. https://doi.org/10.1016/S1050-4648(02)00139-0
  10. Hordvik, I., Thevarajan, J., Samdal, I., Bastani, N. and Krossoy, B. (1999) Molecular cloning and phylogenetic analysis of the Atlantic salmon immunoglobulin D gene. Scand. J. Immunol. 50, 202-210. https://doi.org/10.1046/j.1365-3083.1999.00583.x
  11. Jang, H. N., Woo, J. K., Cho, Y. H., Kyong, S. B. and Choi, S. H. (2004) Characterization of monoclonal antibodies against heavy and light chains of flounder (Paralichthys olivaceus) immunoglobulin. JBMB 47, 52-59
  12. Lee, U. H., Pack, H. J., Do, J. W., Bang, J. D., Cho, H. R., Nam, C. W., Choi, D. H., Yu, H. K., Jeong, C. S., Han, I. S. and Park, J. W. (2001) Flounder (Paralichthys olivaceus) cDNA encoding a secreted immunoglobulin M heavy chain. Fish Shellfish Immunol. 11, 537-540. https://doi.org/10.1006/fsim.2001.0336
  13. Lundgvist, M., Bengtem, E., Stromberg, S. and Pilstrom, L. (1996) Ig light chain gene in the Siberian sturgeon (Acipenser baeri). J. Immunol. 157, 2031-2038.
  14. Magnadottir, B. and Gudmundsdottir, B. K. (1992) A comparison of total and specific immunoglobulin levels in healthy Atlantic salmon (Salmo salar L.) and in salmon naturally infected with Aeromonas salmonicida subsp. Achromogenes. Vet. Immunol. Immunopathol. 32, 179-189. https://doi.org/10.1016/0165-2427(92)90078-5
  15. Miller, N. W., Wilson, M. and Bengten, E. (1998) Functional and molecular characterization of teleost leucocytes. Immunol. Rev. 166, 187-197. https://doi.org/10.1111/j.1600-065X.1998.tb01263.x
  16. Nakao, M., Moritomo, T., Tomana, M., Fujiki, K. and Yano, T. (1998) Isolation of cDNA encoding the constant region of the immunoglobulin heavy chain from common carp (Cyprinus carpio L). Fish shellfish Immunol. 8, 425-434. https://doi.org/10.1006/fsim.1998.0149
  17. Rijkers, G. T., Frederix-wolters, E. M. and van Muiswinkel, W. B. (1980) The immune system of cyprinid fish. Kinetics and temperature dependence of antibody-producing cells in carp (Cyprinus carpio). Immunology 41, 91-97.
  18. Stenvik, J. and Jorgensen, T. O. (2000) Immunoglobulin D (IgD) of Atlantic cod has a unique structure. Immunogenetics 51, 452-461. https://doi.org/10.1007/s002510050644
  19. Thuvander, A., Fossum, C. and Lorenzen, N. (1990) Monoclonal antibodies to salmonid immunoglobulin: characterization and applicability in immunoassays, Dev.Comp. Immunol. 14, 415-423. https://doi.org/10.1016/0145-305X(90)90034-C
  20. Warr, G. W. (1995) The immunoglobulin genes of fish. Dev. Comp. Immunol. 19, 1-12. https://doi.org/10.1016/0145-305X(94)00052-H
  21. Nakao, M., Moritomo, T., Tomana, M., Fujiki, K. and Yano, T. (1998) Isolation of cDNA encoding the constant region of the immunoglobulin heavy chain from common carp (Cyprinus carpio L). Fish shellfish Immunol. 8, 425-434. https://doi.org/10.1006/fsim.1998.0149
  22. Wilson, M., Bengten, E., Miller, N. W., Clem, L. W., du Pasquier, L. and Warr, G. W. (1997) A novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD, Proc. Natl. Acad. Sci. USA 94, 4593-4597. https://doi.org/10.1073/pnas.94.9.4593

Cited by

  1. Common carp have two subclasses of bonyfish specific antibody IgZ showing differential expression in response to infection vol.34, pp.11, 2010, https://doi.org/10.1016/j.dci.2010.06.012
  2. Molecular cloning, tissue distribution and quantitative analysis of two proopiomelanocortin mRNAs in Japanese flounder (Paralichthys olivaceus) vol.42, pp.4, 2009, https://doi.org/10.5483/BMBRep.2009.42.4.206
  3. New insights into the enigma of immunoglobulin D vol.237, pp.1, 2010, https://doi.org/10.1111/j.1600-065X.2010.00929.x
  4. New insights into evolution of IgT genes coming from Antarctic teleosts vol.24, 2015, https://doi.org/10.1016/j.margen.2015.06.009