期刊
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
卷 9, 期 -, 页码 -出版社
FRONTIERS MEDIA SA
DOI: 10.3389/fbioe.2021.792111
关键词
single domain antibody; bamboo shark; IgNAR; immunization; vNAR; immune repertoire
资金
- National Natural Science Foundation of China [61773326, 81770099]
- Hong Kong Health, and Medical Research Fund [05160296]
- Hong Kong Research Grants Council [21101218]
- Shenzhen Science and Technology Innovation Fund [JCYJ20170413115637100, JCYJ20170412152916724]
- Sanming Project of Medicine in Shenzhen [SZSM201811092]
- Tung Biomedical Sciences Centre
The white-spotted bamboo shark has been demonstrated as a promising small animal model for the production of shark single domain antibodies (sdAbs), offering a cost-effective and easy-to-operate alternative to large-scale shark farming. Research has shown that bamboo sharks exhibit effective immune responses and can produce high-affinity IgNAR, paving the way for the discovery and development of sdAbs for the biomedical industry.
The development of shark single domain antibodies (sdAbs) is hindered by the high cost and tediousness of large-sized shark farming. Here, we demonstrated white-spotted bamboo sharks (Chiloscyllium plagiosum) being cultivated commercially as a promising small animal model to produce sdAbs. We found that immunoglobulin new antigen receptor (IgNAR) presented in bamboo shark genome, transcriptome, and plasma. Four complete IgNAR clusters including variable domains (vNARs) were discovered in the germline, and the Variable-Joining pair from IgNAR1 cluster was dominant from immune repertoires in blood. Bamboo sharks developed effective immune responses upon green fluorescent protein (GFP), near-infrared fluorescent protein iRFP713, and Freund's adjuvant immunization revealed by elevated lymphocyte counts and antigen specific IgNAR. Before and after immunization, the complementarity determining region 3 (CDR3) of IgNAR were the major determinant of IgNAR diversity revealed by 400-bp deep sequencing. To prove that bamboo sharks could produce high-affinity IgNAR, we isolated anti-GFP and anti-iRFP713 vNARs with up to 0.3 and 3.8 nM affinities, respectively, from immunized sharks. Moreover, we constructed biparatopic vNARs with the highest known affinities (20.7 pM) to GFP and validated the functions of anti-GFP vNARs as intrabodies in mammalian cells. Taken together, our study will accelerate the discovery and development of bamboo shark sdAbs for biomedical industry at low cost and easy operation.
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