南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (6): 944-948.doi: 10.12122/j.issn.1673-4254.2022.06.20

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腺相关病毒衣壳保守区多价抗原肽的原核表达及多克隆抗体制备

李舒月，曹春雨，张 浩，李玉玲，张雄洲，杨子灿，夏 燕，王 磊，吕亚丰   

  1. 肿瘤微环境与免疫治疗湖北省重点实验室//三峡大学医学院，湖北 宜昌 443000；湖北民族大学附属民大医院风湿性疾病发生与干预湖北省重点实验室，湖北 恩施 445000
  • 出版日期:2022-06-20 发布日期:2022-06-27

Prokaryotic expression of a recombinant protein of adeno-associated virus capsid conserved regions and preparation of its polyclonal antibody

LI Shuyue, CAO Chunyu, ZHANG Hao, LI Yuling, ZHANG Xiongzhou, YANG Zican, XIA Yan, WANG Lei, LÜ Yafeng   

  1. Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, Three Gorges University Medical College, Yichang 443000, China; Hubei Provincial Key Laboratory of Occurrence and Intervention of Rheumatic Diseases, Affiliated Hospital of Hubei University for Nationalities, Enshi 445000, China
  • Online:2022-06-20 Published:2022-06-27

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摘要： 目的 原核表达和纯化腺相关病毒（AAV）衣壳保守区抗原肽，制备抗AAV衣壳保守区的兔多克隆抗体。方法 设计并合成编码AAV衣壳蛋白保守区的DNA，将序列克隆到载体pET30a，获得质粒pET30a-AAV-CR，原核表达并纯化保守区多价抗原肽，考马斯蓝染色法及Western blot法对AAV保守区多价抗原肽进行鉴定。将日本大耳朵白兔分为实验组与对照组（1只/组），实验组注射AAV保守区蛋白制备多克隆抗体，对照组注射PBS，ELISA检测抗体效价，Western blot及细胞免疫荧光法检测抗体应用效果。结果 成功构建了表达AAV衣壳保守区抗原肽的质粒pET30a-AAV-CR，表达纯化得到相对分子质量为17000的蛋白质；纯化后的蛋白可在兔体内诱导产生针对AAV衣壳保守区的抗体，且该抗体能广泛性识别AAV1-AAV10衣壳蛋白序列。结论 诱导出AAV衣壳保守区蛋白并成功获得了抗AAV衣壳保守区的多克隆抗体，为后续载体开发以及生物学功能研究奠定了基础。

关键词: 腺相关病毒；多价抗原肽；蛋白纯化；多克隆抗体

Abstract: Objective To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody. Methods The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay. Results The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10. Conclusion We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.

Key words: adeno-associated virus; polyvalent antigenic peptide; protein purification; polyclonal antibodies