Screening and identification of nanobody against inhibin α-subunit from a Camelus bactrianus phage display library

Background: Inhibin is a member of the transforming growth factor family that influences reproduction in animals. Objective: The purpose of this study was to obtain nanobodies from the phage antibody library constructed by us that can specifically bind to inhibin alpha-subunit. Methods: In this study, camels were immunized with Kazakh sheep inhibin-alpha protein that expressed in BL21 E. coli, and the camel VHH nanobody phage display library was prepared using nested PCR. The nanobodies specifically binding to inhibin alpha-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. Results: A nanobody display library was successfully constructed with a capacity of 1.05 x 10(12 )CFU, and four inhibin-alpha-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 complexed with inhibin-alpha protein mainly through hydrophobic bonds. Immunity tests revealed that the nanobody Nb-4 could effectively inhibit sheep inhibin A/B and could significantly improve the FSH level in sheep. Conclusion: Four inhibin alpha-subunit-specific nanobodies with biological functions were successfully screened. To the best of our knowledge, this is a new reproductive immunomodulatory pathway of inhibin alpha-subunit, which may change the secretion of FSH in the ovary, thus changing the estrous cycle of organisms.