抑制素α亚基特异性纳米抗体基因筛选与鉴定

doi:10.16431/j.cnki.1671-7236.2023.04.025

Abstract

Abstract: 【Objective】 The aim of this study was to screen the inhibin α subunit (INHα)-specific nanobody (VHH) gene from the lymphocyte genome of Xingjiang Bactrian camel, and prepare a novel inhibin immune preparation,so as to indirectly increase the blood follicle-stimulating hormone (FSH) level and ovulation rate and lambing rate in animal.【Method】 INHα protein was induced by pET32a-INHA prokaryotic expression vector and purified.The purified INHα protein was renatured with urea solution and then immunized Xinjiang Bactrian camels.The VHH amino acid difference database was established by nested PCR amplification of lymphocyte VHH gene in whole blood before and after immunization and high-throughput sequencing.The pre-immune serum,the post-immune serum and the INHα-specific antibodies screened from the post-immunization serum were analyzed by liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) to obtain mass spectrometry data.After performing protein database search and data analysis on mass spectrometry data,a peptide library and protein library of INHα-specific antibodies and post-immunization-specific antibodies were established.By aligning the VHH amino acid difference database with the peptide library of INHα-specific antibody and post-immunization-specific antibody,respectively,the INHα-specific nanobody gene was screened out.Finally,the screened VHH genes were analyzed by sequence alignment,three-dimensional structure prediction and protein-protein simulation docking.【Result】 INHα protein was successfully expressed and purified,and the antibody titer of Xinjiang Bactrian camel immunized with INHα protein reached 1∶1 024 000.The VHH gene was successfully cloned from the lymphocytes,and the specific antibody against INHα was screened from the immunized serum.Finally,5 INHα-specific nanobody genes were screened by high-throughput sequencing,mass spectrometry analysis and data processing.The amino acid sequence alignment of the 5 strains of VHH showed that the overall similarity was 70.98%,and the length of the complementarity determining region 1 (CDR1) ranged from 10 to 13,the length of CDR2 ranges from 12 to 13,the length of CDR3 ranges from 12 to 22,and CDR3 had the largest amino acid sequence difference among the three complementarity determining regions.Phylogenetic tree showed that the selected 5 amino acid sequences had rich diversity.The results of protein-protein docking simulation showed that the other 4 VHH strains could pair with INHα except for VHH-267,and the main forces were hydrophobic interaction and hydrogen bonding.【Conclusion】 In this study,5 strains of INHα-specific VHH genes were firstly screened from the lymphocyte genome of Xinjiang Bactrian camel by high-throughput sequencing technology combined with mass spectrometry.This study would provide theoretical guidance and technical support for improving the level of FSH and ovulation rate in animals,and had certain reference value for the development of reproductive immunology.

Key words: inhibin α subunit (INHα); Bactrian camel; nanobody; high throughput sequencing; mass spectrometry analysis

CLC Number:

S852.4^＋3

MA Jifu, BAKHET·Bodai, KEZERBEK·Kazibek, LI Zhenwei, ORALHAZI·Hazikhan. Screening and Identification of Inhibin α Subunit-specific Nanobody Gene[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1532-1542.

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Screening and Identification of Inhibin α Subunit-specific Nanobody Gene

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