双峰驼Cyp-A的原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.07.029

摘要/Abstract

摘要： 【目的】制备双峰驼亲环素A （cyclophylin A，Cyp-A）的多克隆抗体，为揭示双峰驼体内的黏膜免疫反应机理提供依据。【方法】从GenBank数据库中双峰驼Cyp-A基因序列（登录号：XM_010969543.1）上选取编码区，截取不含信号肽的膜外序列进行碱基优化后，与pET-28a （+）载体连接构建重组质粒pET-28a-Cyp-A。将重组质粒转化大肠杆菌BL21（DE3）感受态细胞，用IPTG诱导表达并对表达条件进行优化，优化诱导条件后通过SDS-PAGE鉴定重组蛋白的表达情况。纯化重组蛋白并免疫家兔，制备兔抗双峰驼Cyp-A多克隆抗体，分别使用ELISA和Western blotting检测抗体效价及特异性，并采用HE和SABC免疫组化染色法对Cyp-A多克隆抗体进行定位观察。【结果】重组质粒pET-28a-Cyp-A主要以包涵体形式表达，Cyp-A蛋白约为17 ku，最佳诱导条件为0.7 mmol/L IPTG诱导8 h，杂蛋白最佳洗脱液为5 mmol/L咪唑。经ELISA和Western blotting检测，兔抗双峰驼Cyp-A多克隆抗体的效价为1：64 000，能特异性识别重组蛋白Cyp-A。经HE和SABC免疫组化染色观察，Cyp-A多克隆抗体仅能使回肠滤泡相关上皮（FAE）中的M细胞特异性着色。【结论】试验成功制备出效价高、特异性强的兔抗双峰驼Cyp-A多克隆抗体，能特异性识别双峰驼回肠中M细胞。

关键词: 双峰驼; 亲环素A(Cyp-A); 原核表达; 多克隆抗体; 定位

Abstract: 【Objective】 The polyclonal antibody of cyclophilin A (Cyp-A) was prepared, in order to provide a basis for revealing the mechanism of mucosal immune response in Bactrian camel.【Method】 The coding region was selected from the Cyp-A gene sequence of Bactrian camel in GenBank (accession No.:XM_010969543.1).The extracellular sequence without signal peptide was intercepted and optimized its base, and the recombinant plasmid pET-28a-Cyp-A was constructed by connecting it with pET-28a (+) vector.The recombinant plasmid pET-28a-Cyp-A was transformed into E.coli BL21 (DE3) competent cell to induce expression.After optimizing the induction conditions, the expression of the recombinant protein was identified by SDS-PAGE.Then the recombinant protein was purified and used to immunize rabbits to prepare rabbit anti-Bactrian camel Cyp-A polyclonal antibody.The antibody titer and specificity were detected by ELISA and Western blotting, respectively.HE and SABC immunohistochemical staining methods were used to observe the location of Cyp-A using self-made polyclonal antibody.【Result】 The recombinant plasmid pET-28a-Cyp-A was mainly expressed in the form of inclusion body.The size of Cyp-A protein was about 17 ku.The optimal induction conditions were 0.7 mmol/L IPTG and 8 h.The optimal elution concentration of the protein was 5 mmol/L imidazole.By ELISA and Western blotting detection, the titer of rabbit anti-Bactrian camel Cyp-A polyclonal antibody was 1:64 000, which could specifically recognize the recombinant protein Cyp-A.HE and SABC immunohistochemical staining results showed that Cyp-A polyclonal antibody could only specifically stain M cells in ileum follicular associated epithelium (FAE).【Conclusion】 The rabbit anti-Bactrian camel Cyp-A polyclonal antibody with high titer and specificity was successfully prepared, which could be used to specifically recognize M cells in ileum of Bactrian camel.

Key words: Bactrian camel; cyclophilin A (Cyp-A); prokaryotic expression; polyclonal antibody; location

中图分类号:

S824

索南吉, 李沛轩, 刘转霞, 王亭玮, 刘柯江, 谢东旭, 曾巍巍, 王雯慧. 双峰驼Cyp-A的原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(7): 2896-2905.

SUO Nanji, LI Peixuan, LIU Zhuanxia, WANG Tingwei, LIU Kejiang, XIE Dongxu, ZENG Weiwei, WANG Wenhui. Prokaryotic Expression and Polyclonal Antibody Preparation of Cyp-A in Bactrian Camel[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2896-2905.

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