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

摘要/Abstract

摘要： 【目的】从新疆双峰驼淋巴细胞基因组中筛选出抑制素α亚基(INHα)特异性纳米抗体(VHH)基因,制备新型抑制素免疫制剂,以期间接提高动物血液促卵泡素(FSH)水平、排卵率和产羔率。【方法】利用pET32a-INHA原核表达载体诱导表达INHα蛋白并纯化,纯化后的INHα蛋白经尿素溶液复性后免疫新疆双峰驼。通过对免疫前和免疫后全血中淋巴细胞VHH基因进行巢式PCR扩增、高通量测序并建立VHH氨基酸差异数据库;分别对免疫前和免疫后血清及从免疫后血清中筛选出的INHα特异性抗体进行液相色谱质谱/质谱联用(LC-MS/MS)分析获得质谱数据;对质谱数据进行蛋白数据库检索和数据分析后建立INHα特异性抗体和免疫后特异性抗体的肽段库和蛋白库;通过将VHH氨基酸差异数据库分别与INHα特异性抗体和免疫后特异性抗体的肽段库进行序列比对,筛选出INHα特异性纳米抗体基因;对筛选出的VHH基因进行序列比对、三维结构预测和蛋白-蛋白模拟对接。【结果】试验成功诱导表达并纯化了INHα蛋白,INHα蛋白免疫新疆双峰驼获得的抗体效价达1∶1024000;成功从淋巴细胞中克隆出VHH基因,从免疫后血清中筛选出INHα特异性抗体;通过高通量测序、质谱分析及数据处理筛选出5个INHα特异性VHH基因,其氨基酸序列比对结果显示,整体相似性为70.98%,其中互补决定区1(CDR1)长10~13个氨基酸,CDR2长12~13个氨基酸,CDR3长12~22个氨基酸,CDR3在3个互补决定区中氨基酸序列差异最大;系统进化树显示,选择的5条氨基酸序列具有丰富的多样性;蛋白-蛋白模拟对接结果表明,除VHH-267外其余4株纳米抗体均可与INHα配对连接,且主要作用力为疏水相互作用和氢键。【结论】本研究首次通过高通量测序技术联合质谱分析从新疆双峰驼淋巴细胞基因组中筛选出5个INHα特异性VHH基因。本研究结果可为提高动物机体FSH水平及排卵率提供理论指导和技术支持,对繁殖免疫学的发展具有一定参考价值。

关键词: 抑制素α亚基(INHα); 双峰驼; 纳米抗体; 高通量测序; 质谱分析

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

中图分类号:

S852.4^＋3

马继福, 巴合提·博代, 黑子别克·哈孜别克, 李振伟, 吾热力哈孜·哈孜汗. 抑制素α亚基特异性纳米抗体基因筛选与鉴定[J]. 中国畜牧兽医, 2023, 50(4): 1532-1542.

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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