VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica)

Frontiers in Bioengineering and Biotechnology

Bioengineering and Biotechnology

Tatsuhiko Ozawa¹  Yoh Ohnuki²  Honoka Tomoda³  Akihiko Takechi³  Takaaki Shimizu³  Kimiyoshi Narita³  Takuya Miyakawa⁴  Yuji Ito⁵  Wei Zhou⁶  Hiroki Zenke⁶  Tatsuya Sawasaki⁶  Hiroyuki Takeda⁶  Yuri Umeda⁶ 

[1] Department of Immunology, Faculty of Medicine, Academic Assembly, University of Toyama, Toyama, Japan;Center for Advanced Antibody Drug Development, University of Toyama, Toyama, Japan;Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan;Fisheries Research Center, Ehime Research Institute of Agriculture, Forestry and Fisheries, Iyo, Japan;Graduate School of Biostudies, Kyoto University, Kyoto, Japan;Graduate School of Science and Engineering, Kagoshima University, Kagoshima, Japan;Proteo-Science Center, Ehime University, Matsuyama, Japan;

关键词: VNAR;    Japanese topeshark;    phage display;    yeast display;    biopanning;    deep sequencing;   

DOI  :  10.3389/fbioe.2023.1265582

 received in 2023-07-23, accepted in 2023-08-28,  发布年份 2023

来源: Frontiers

【 摘 要 】

The VNAR (Variable New Antigen Receptor) is the smallest single-domain antibody derived from the variable domain of IgNAR of cartilaginous fishes. Despite its biomedical and diagnostic potential, research on VNAR has been limited due to the difficulties in obtaining and maintaining immune animals and the lack of research tools. In this study, we investigated the Japanese topeshark as a promising immune animal for the development of VNAR. This shark is an underutilized fishery resource readily available in East Asia coastal waters and can be safely handled without sharp teeth or venomous stingers. The administration of Venus fluorescent protein to Japanese topesharks markedly increased antigen-specific IgM and IgNAR antibodies in the blood. Both the phage-display library and the yeast-display library were constructed using RNA from immunized shark splenocytes. Each library was enriched by biopanning, and multiple antigen-specific VNARs were acquired. The obtained antibodies had affinities of 1 × 10−8 M order and showed high plasticity, retaining their binding activity even after high-temperature or reducing-agent treatment. The dissociation rate of a low-affinity VNAR was significantly improved via dimerization. These results demonstrate the potential utility of the Japanese topeshark for the development of VNAR. Furthermore, we conducted deep sequencing analysis to reveal the quantitative changes in the CDR3-coding sequences, revealing distinct enrichment bias between libraries. VNARs that were primarily enriched in the phage display had CDR3 coding sequences with fewer E. coli rare codons, suggesting translation machinery on the selection and enrichment process during biopanning.

【 授权许可】

Unknown   
Copyright © 2023 Takeda, Ozawa, Zenke, Ohnuki, Umeda, Zhou, Tomoda, Takechi, Narita, Shimizu, Miyakawa, Ito and Sawasaki.