Construction of an artificially randomized IgNAR phage display library: screening of variable regions that bind to hen egg white lysozyme.

Marine Biotechnology
Maki OhtaniTakashi Aoki

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 × 10(7) 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 ra...Continue Reading

References

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Aug 7, 2010·Fish & Shellfish Immunology·Yuka HondaTakashi Aoki

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Citations

Mar 19, 2014·Current Microbiology·Walead Ebrahimizadeh, Masoumeh Rajabibazl
Aug 5, 2014·Expert Opinion on Biological Therapy·Marina KovalevaCaroline Barelle
Mar 29, 2014·Molecular Immunology·Jinny L LiuEllen R Goldman
Mar 9, 2017·Protein Science : a Publication of the Protein Society·Thomas Böldicke
Oct 19, 2017·Diagnostics·Chiuan Herng LeowJames McCarthy
Apr 11, 2018·Journal of Preventive Medicine and Public Health = Yebang Ŭihakhoe Chi·Do-Hyeon ParkHee Young Shin
Aug 31, 2019·Molecular Biotechnology·Rose H ReaderKevin C Gough
Mar 10, 2021·Current Microbiology·Taruna AnandBhupendra N Tripathi

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