China Animal Husbandry and Veterinary Medicine ›› 2023, Vol. 50 ›› Issue (5): 2032-2043.doi: 10.16431/j.cnki.1671-7236.2023.05.030

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Screening of Nanobody Specific to Inhibin α Subunit Based on Phage Display Technology

MA Jifu1, Adil·Ketan2, XIE Jingang1, Bakhet·Bodai1, LI Zhenwei1, Oralhazi·Hazikhan1   

  1. 1. College of Animal Science and Technology, Shihezi University, Shihezi 832000, China;
    2. Xinjiang Tacheng District Animal Disease Control and Diagnosis Center, Tacheng 834700, China
  • Revised:2023-02-21 Online:2023-05-05 Published:2023-04-28

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Abstract: 【Objective】 The anti-inhibin α subunit (INHα) nanobody library was constructed by phage display technology,and the specific nanobodies against sheep INHα were screened, so as to prepare for the improvement of peripheral blood follicle-stimulating hormone (FSH) level and ovulation rate in sheep.【Method】 The expression of INHα protein was induced by the pET32a-INHA prokaryotic expression vector preserved in the laboratory,and the induced INHα protein was identified by SDS-PAGE and Western blotting.Then the purified INHα protein was used to immunize Xinjiang bactrian camel.The camel phage display nanobody library was prepared by nested PCR amplification.The specific nanobody against INHα was enriched and screened from the library by three rounds of immunoaffinity screening and phage ELISA.The gene sequence of the nanobody was obtained by sequencing. The affinity of the nanobody to INHα protein was preliminarily identified by protein-protein analog docking.【Result】 The results showed that the recombinant protein of INHα with a size of 36 ku was obtained by prokaryotic induction expression and Ni agarose purification,and a protein band of about 36 ku was detected by Western blotting.Bactrian camel immunized with INHα protein for 6 times and the antibody titer reached 1∶1 024 000.The VHH gene of about 400 bp was obtained by nested PCR,and a nanobody library with a capacity of 1.05×1012CFU was constructed by electrotransformation and other experiments.The positive rate of the library was 94%.The anti-INHα nanobody library of 3.0×108 CFU was obtained by three rounds of immunoaffinity screening.Phage ELISA was used to obtain four strains of INHα-specific nanobodies (VHH-4,VHH-29,VHH-89 and VHH-108) and the gene sequence similarity of the four INHα-specific nanobodies was 80.31%.Through protein-protein simulation docking,it was found that all the four nanobodies had strong affinity with INHα.In addition,the total free energy of VHH-4 and INHα was -576.28 kJ/mol,which was the best antibody among the four VHH.【Conclusion】 An anti-INHα nanobody library was successfully constructed,and four INHα-specific nanobodies,VHH-4,VHH-29,VHH-89 and VHH-108,were obtained from the library.The results of this study could provide reference for the application of nanobodies in reproductive immunology.

Key words: inhibin α subunit (INHα); nanobody; phage display technology

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