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Combined techniques for antigen-specific nanobody discovery in the white-spotted bamboo shark (Chiloscyllium
Bo Zhong1, Junjie Yang2, Kaixi Zheng2
1College of Life Sciences and Medicine, Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Sci-Tech University, 310018, Hangzhou, China; School of Life Sciences, Central South University, 410031, Changsha, China.
Antibodies are pivotal molecules in the immune system, playing crucial roles in disease diagnosis, treatment, and research. Among these, shark-derived nanobody (VNAR) antibodies stand out as the smallest known antigen-binding fragments, with a molecular weight of approximately 12 kDa. Their unique size allows for efficient targeting of antigens, making them valuable tools in biomedicine. However, traditional phage display assays for screening shark nanobodies have limitations, as not all nanobodies express effectively in this format. In this study, we innovatively employed Cre recombinase as an antigen and integrated next-generation sequencing, cell sorting, phage display, and mass spectrometry to enhance the screening of antigen-specific shark nanobodies. Through diverse screening strategies, we successfully identified a substantial array of VNARs. Functional assays in mammalian cells confirmed that these VNARs bind specifically to Cre recombinase without inhibiting its enzymatic activity. Our findings not only advance the methodologies for VNAR screening but also open new avenues for the development of targeted therapeutics and immunological research in aquatic fish.
Antibodies are pivotal molecules in the immune system, playing crucial roles in disease diagnosis, treatment, and research. Among these, shark-derived nanobody (VNAR) antibodies stand out as the smallest known antigen-binding fragments, with a molecular weight of approximately 12 kDa. Their unique size allows for efficient targeting of antigens, making them valuable tools in biomedicine. However, traditional phage display assays for screening shark nanobodies have limitations, as not all nanobodies express effectively in this format. In this study, we innovatively employed Cre recombinase as an antigen and integrated next-generation sequencing, cell sorting, phage display, and mass spectrometry to enhance the screening of antigen-specific shark nanobodies. Through diverse screening strategies, we successfully identified a substantial array of VNARs. Functional assays in mammalian cells confirmed that these VNARs bind specifically to Cre recombinase without inhibiting its enzymatic activity. Our findings not only advance the methodologies for VNAR screening but also open new avenues for the development of targeted therapeutics and immunological research in aquatic fish.
