Screening and Preliminary Identification of Inhibin α Subunit-Specific Nanobodies by High-Throughput Sequencing Combined with Mass Spectrometry

Abstract

Abstract Inhibin is mainly a glycoprotein heterodimer secreted by female ovaries and male testes, which belongs to the TGF-β superfamily. In female animals, inhibin inhibits pituitary follicle stimulating hormone (FSH) synthesis through the endocrine pathway, and regulates follicular development, gametogenesis, and hormone secretion. In this study, high-throughput sequencing of the nanobody (VHH）gene in lymphocytes of Bactrian camels before and after immunization with inhibin α protein and mass spectrometry analysis of specific antibodies to inhibin α protein in serum after immunization were used to screen for inhibin α subunit-specific nanobodies. The results of high-throughput sequencing showed that there were 57841 valid sequences in the VHH database before immunization, 53994 in the VHH database after immunization, and 816 in the specific VHH database after immunization. After searching the database, the results of mass spectrometry showed that 35 peptides and 135 proteins were found in the serum-specific antibodies after immunization. Inhibin α subunit-specific antibody contains 31 peptides and 33 proteins. Finally, 10 nanobody gene sequences were screened according to the location of the complementary determinant region and protein score, namely Nb-1712, Nb-573, Nb-267, Nb-1971, Nb-2000, Nb-799, Nb-1581, Nb-2004, Nb-1737, and Nb-338. In addition, 10 nanobodies had high affinity to the inhibin α protein by protein simulation docking and indirect enzyme linked immunosorbent assay (ELISA ) affinity identification. In this study, 10 inhibin α subunit-specific nanobody genes were screened from the lymphocyte genome of a Xinjiang Bactrian camel by high-throughput sequencing combined with mass spectrometry for the first time, and their affinity with the inhibin α subunit was preliminary identified. This study will provide theoretical guidance and technical support for improving the FSH level and ovulation rate of animals and will also provide a certain reference value for the development of reproductive immunology.