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 α-subunit. Methods: In this study, camels were immunized with Kazakh sheep inhibin-α 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 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. Results: 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 complexed with inhibin-α 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 α-subunit-specific nanobodies with biological functions were successfully screened. 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.

Genetic Diversity of Hard Ticks (Acari: Ixodidae) in the South and East Regions of Kazakhstan and Northwestern China

To date, there is no report on the genetic diversity of ticks in these regions. A total of 370 representative ticks from the south and east regions of Kazakhstan (SERK) and Xinjiang Uygur Autonomous Region (XUAR) were selected for molecular comparison. A fragment of the mitochondrial cytochrome c oxidase subunit I (cox1) gene, ranging from 631 bp to 889 bp, was used to analyze genetic diversity among these ticks. Phylogenetic analyses indicated 7 tick species including Hyalomma asiaticum, Hyalomma detritum, Hyalomma anatolicum, Dermacentor marginatus, Rhipicephalus sanguineus, Rhipicephalus turanicus and Haemaphysalis erinacei from the SERK clustered together with conspecific ticks from the XUAR. The network diagram of haplotypes showed that i) Hy. asiaticum from Almaty and Kyzylorda Oblasts together with that from Yuli County of XUAR constituted haplogroup H-2, and the lineage from Chimkent City of South Kazakhstan was newly evolved; and ii) the R. turanicus ticks sampled in Israel, Almaty, South Kazakhstan, Usu City, Ulugqat and Baicheng Counties of XUAR were derivated from an old lineage in Alataw City of XUAR. These findings indicate that: i) Hy. asiaticum, R. turanicus and Ha. erinacei shared genetic similarities between the SERK and XUAR; and ii) Hy. marginatum and D. reticulatus show differences in their evolution.