Abstract
To develop a multi-antigen-specific immunoglobulin new antigen receptor (IgNAR) variable (V) region phage display library, CDR3 in the V region of IgNAR from banded houndshark (Triakis scyllium) was artificially randomized, and clones specific for hen egg white lysozyme (HEL) were obtained by the biopanning method. The nucleotide sequence of CDR3 in the V region was randomly rearranged by PCR. Randomized CDR3-containing segments of the V region were ligated into T7 phage vector to construct a phage display library and resulted in a phage titer of 3.7 × 107 PFU/ml. Forty clones that contained randomized CDR3 inserts were sequenced and shown to have different nucleotide sequences. The HEL-specific clones were screened by biopanning using HEL-coated ELISA plates. After six rounds of screening, nine clones were identified as HEL-specific, eight of which showed a strong affinity to HEL in ELISA compared to a negative control (i.e., empty phage clone). The deduced amino acid sequences of CDR3 from the HEL-specific phage clones fell into four types (I−IV): type I contains a single cysteine residue and type II−IV contain two cysteine residues. These results indicated that the artificially randomized IgNAR library is useful for the rapid isolation of antigen-specific IgNAR V region without immunization of target antigen and showed that it is possible to isolate an antigen-specific IgNAR V region from this library.
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References
Aitken R (2009) Antibody phage display: methods and protocols, 2nd edn. Humana press, New York
Casson LP, Manser T (1995) Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen. J Exp Med 182:743–750
Diaz M, Greenberg AS, Flajnik MF (1998) Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: possible role in antigen-driven reactions in the absence of germinal centers. Proc Natl Acad Sci USA 95:14343–14348
Dolk E, van Vliet C, Perez JM, Vriend G, Darbon H, Ferrat G, Cambillau C, Frenken LG, Verrips T (2005) Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen. Proteins 59:555–564
Dooley H, Flajnik MF, Porter AJ (2003) Selection and characterization of naturally occurring single-domain (IgNAR) antibody fragments from immunized sharks by phage display. Mol Immunol 40:25–33
Dooley H, Flajnik MF (2005) Shark immunity bites back: affinity maturation and memory response in the nurse shark, Ginglymostoma cirratum. Eur J Immunol 35:936–945
Dooley H, Flajnik MF (2006) Antibody repertoire development in cartilaginous fish. Dev Comp Immunol 30:43–56
Greenberg AS, Avila D, Hughes M, Hughes A, McKinney EC, Flajnik MF (1995) A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374:168–173
Honda Y, Kondo H, Caipang CM, Hirono I, Aoki T (2010) cDNA cloning of the immunoglobulin heavy chain genes in banded houndshark Triakis scyllium. Fish Shellfish Immunol 29:854–861
Kobayashi N, Kato Y, Oyama H, Taga S, Niwa T, Sun P, Ohtoyo M, Goto J (2008) Anti-estradiol-17β single-chain Fv fragments: generation, characterization, gene randomization, and optimized phage display. Steroids 73:1485–1499
Liu JL, Anderson GP, Delehanty JB, Baumann R, Hayhurst A, Goldman ER (2007) Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library. Mol Immunol 44:1755–17783
Muyldermans S, Cambillau C, Wyns L (2001) Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem Sci 26:230–235
Nuttall SD, Krishnan UV, Doughty L, Nathanielsz A, Ally N, Pike RN, Hudson PJ, Kortt AA, Irving RA (2002) A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis. FEBS Lett 516:80–86
Nuttall SD, Krishnan UV, Hattarki M, De Gori R, Irving RA, Hudson PJ (2001) Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries. Mol Immunol 38:313–326
Roux KH, Greenberg AS, Greene L, Strelets L, Avila D, McKinney EC, Flajnik MF (1998) Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins. Proc Natl Acad Sci USA 95:11804–11809
Shao CY, Secombes CJ, Porter AJ (2007) Rapid isolation of IgNAR variable single-domain antibody fragments from a shark synthetic library. Mol Immunol 44:656–665
Saerens D, Frederix F, Reekmans G, Conrath K, Jans K, Brys L, Huang L, Bosmans E, Maes G, Borghs G, Muyldermans S (2005) Engineering camel single-domain antibodies and immobilization chemistry for human prostate-specific antigen sensing. Anal Chem 77:7547–7555
Stanfield RL, Dooley H, Flajnik MF, Wilson IA (2004) Crystal structure of a shark single-domain antibody V region in complex with lysozyme. Science 305:1770–1773
Acknowledgments
We thank Dr. Yoshisuke Nishi (Nagahama Institute of Bio-Science and Technology) for advising in the construction of phage display library. This work was supported by grant-in-aid for scientific research [KAKENHI KIBAN KENKYU (A) 21248025] from the Ministry of Education, Culture, Sports, Science and Technology, Japan. This research was also supported by a grant from the World Class University Program (R32-10253), funded by the Korean Ministry of Education, Science, and Technology of South Korea.
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Ohtani, M., Hikima, Ji., Jung, T.S. et al. Construction of an Artificially Randomized IgNAR Phage Display Library: Screening of Variable Regions that Bind to Hen Egg White Lysozyme. Mar Biotechnol 15, 56–62 (2013). https://doi.org/10.1007/s10126-012-9456-1
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DOI: https://doi.org/10.1007/s10126-012-9456-1