Construction and next-generation sequencing analysis of a large phage-displayed VNARsingle-domain antibody library from six naïve nurse sharks
PublisherOxford University Press
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AbstractBackground: Shark new antigen receptor variable domain (VNAR) antibodies can bind restricted epitopes that may be inaccessible to conventional antibodies. Methods: Here, we developed a library construction method based on polymerase chain reaction (PCR)Extension Assembly and Self-Ligation (named “EASeL”) to construct a large VNARantibody library with a size of 1.2 × 1010from six naïve adult nurse sharks (Ginglymostoma cirratum). Results: The next-generation sequencing analysis of 1.19 million full-length VNARs revealed that this library is highly diversified because it covers all four classical VNARtypes (Types I–IV) including 11% of classical Type I and 57% of classical Type II. About 30% of the total VNARs could not be categorized as any of the classical types. The high variability of complementarity determining region (CDR) 3 length and cysteine numbers are important for the diversity of VNARs. To validate the use of the shark VNARlibrary for antibody discovery, we isolated a panel of VNARphage binders to cancer therapy-related antigens, including glypican-3, human epidermal growth factor receptor 2 (HER2), and programmed cell death-1 (PD1). Additionally, we identified binders to viral antigens that included the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) spike proteins. The isolated shark single-domain antibodies including Type I and Type II VNARs were produced in Escherichia coli and validated for their antigen binding. A Type II VNAR(PE38-B6) has a high affinity (Kd= 10.1 nM) for its antigen. Conclusions: The naïve nurse shark VNARlibrary is a useful source for isolating single-domain antibodies to a wide range of antigens. The EASeL method may be applicable to the construction of other large diversity gene expression libraries. Copyright The Author(s) 2018.
KeywordGene library construction
Middle East Respiratory Syndrome Coronavirus
Identifier to cite or link to this itemhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85071516744&doi=10.1093%2fabt%2ftby011&partnerID=40&md5=03a0372921bddd2f01acf64ba422e5d7; http://hdl.handle.net/10713/12445