高效价猪圆环病毒Ⅱ型Cap蛋白多克隆抗体的制备及应用

doi:10.13560/j.cnki.biotech.bull.1985.2019-1209

摘要/Abstract

摘要： 猪圆环病毒（Porcine circovirus,PCV）衣壳蛋白（Capsid protein,Cap）是病毒主要的抗原蛋白,在病毒感染和宿主免疫应答中起重要作用。为了制备抗PCV2b Cap蛋白的高效价多克隆抗体,利用大肠杆菌表达系统对不含核定位信号肽的PCV2b Cap进行了原核表达,获得了可溶性重组蛋白。将纯化后的重组蛋白免疫新西兰白兔,制备抗血清,并进一步以辛酸-硫酸铵沉淀法提纯IgG。ELISA检测显示所制备抗体效价可达到1∶10⁷,表明抗体具有较高效价。应用该抗体对人工感染PCV2b的PK-15细胞、小鼠组织进行了免疫印迹、间接免疫荧光和免疫组织化学检测,结果表明该抗体在多种检测方法中均能与目的抗原发生特异性结合。该研究制备的高效价抗体为PCV2b的临床检测及科学研究提供了候选工具。

关键词: 猪圆环病毒II型, 衣壳蛋白, 原核表达, 多克隆抗体, 病毒检测

Abstract: Capsid protein（Cap）is the major antigen protein of porcine circovirus（PCV）and plays critical roles in viral infection and elicitation of host immune responses. To prepare a high-titer polyclonal antibody against PCV2b Cap,a PCV2b Cap truncated protein without nuclear localization signal peptide was expressed by Escherichia coli prokaryotic expression system,and soluble recombinant protein was acquired. White rabbits were then immunized with the purified soluble recombinant protein to generate antiserum and anti-Cap IgG was further purified by caprylic acid-ammonium sulfate precipitation. ELISA analysis showed that the titer of the purified anti-Cap antibody was 1∶10⁷,indicating that the antibody was really in high-titer. The antibody was then applied to infect PCV2b-infected PK-15 cells or mouse tissues as well as clinical samples,and then they were detected by immunoblotting,indirect immunofluorescence,or immunohistochemistry. The results showed that the antibody specifically bound with target antigens. In summary,the prepared high-tier anti-Cap polyclonal antibody in this study may provide a candidate tool for the diagnosis and research of PCV2b-assciated diseases.

Key words: porcine circovirus type 2, capsid protein, prokaryotic expression, polyclonal antibody, virus detection

张文, 刘照贞, 李俊硕, 商营利. 高效价猪圆环病毒Ⅱ型Cap蛋白多克隆抗体的制备及应用[J]. 生物技术通报, 2020, 36(7): 72-79.

ZHANG Wen, LIU Zhao-zhen, LI Jun-shuo, SHANG Ying-li. Preparation and Potential Application of High-titer Polyclonal Antibody Against Capsid Protein of Porcine Circovirus Type Ⅱ[J]. Biotechnology Bulletin, 2020, 36(7): 72-79.

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