Screening and Identification of Nanobody against Inhibin α-Subunit from a Camelus Bactrianus Phage Display Library

Abstract

Abstract The inhibin α-subunit is a member of the transforming growth factor family that influences animal reproduction. The purpose of this study was to obtain nanobodies from the phage antibody library constructed by us that can specifically bind to inhibin α-subunit. Such nanobodies can indirectly increase the level of follicle stimulating hormone (FSH) and promote follicular maturation. In this study, Xinjiang Bactrian camel was immunized with prokaryote-expressed inhibin α protein, and the camel phage nanobody display library was prepared by nest polymerase chain reaction (PCR). The nanobodies specifically binding to inhibin α-subunit in the library were screened by three rounds of immunoaffinity screening and phage enzyme-linked immunosorbent assay (phage ELISA). The functions of the selected nanobodies were identified using molecular simulation docking, ELISA affinity test, and sheep immunity test. The results revealed that a nanobody display library was successfully constructed with a capacity of 1.05 × 1012 CFU, and four inhibin-α-subunit-specific nanobodies with an overall similarity of 69.34% were screened from the library, namely, Nb-4, Nb-15, Nb-26, and Nb-57. The results of molecular simulation docking revealed that four types of nanobodies were linked to inhibin-α protein mainly through hydrophobic bonds. Immune tests revealed that the immunity of nanobody Nb-4 could effectively inhibit the level of sheep inhibin A/B, and the nanobody Nb-4 could significantly improve the FSH level in sheep. In summary, four nanobodies with biological function were successfully screened from the constructed antiinhibin α-subunit nanobody library using phage display technique. To the best of our knowledge, this is a new reproductive immunomodulatory pathway of inhibin α-subunit, which may change the secretion of FSH in the ovary, thus changing the estrous cycle of organisms. This study provided a reference for the application of nanobodies in reproductive immunology.