猪肺炎支原体P97蛋白单克隆抗体的制备、表位鉴定及其初步应用

doi:10.11843/j.issn.0366-6964.2020.05.022

摘要/Abstract

摘要： 为了制备能够用于阻断ELISA检测的猪肺炎支原体（Mycoplasma hyopneumoniae，Mhp）单克隆抗体（MAb），本研究将原核表达的Mhp J株P97蛋白C末端包含R1区的肽段（P97CR1）作为免疫原免疫BALB/C雌鼠，筛选获得一株能稳定分泌抗Mhp P97蛋白MAb的杂交瘤细胞株A3。鉴定结果显示，A3 MAb是IgG1亚类，轻链为κ链。Western blot结果显示A3 MAb能够和原核表达纯化的P97CR1蛋白特异性反应，并且在流式细胞仪分析中能够识别天然的Mhp。通过逐渐截短表达分析该MAb识别的表位序列为LDDNLQ，该抗原表位在Mhp菌株中高度保守。阻断ELISA初步试验结果显示，A3 MAb与蛋白抗原的结合能够被Mhp高免阳性血清阻断。该MAb的制备为进一步建立特异性强、灵敏度高的Mhp阻断ELISA检测方法奠定了基础。

关键词: 猪肺炎支原体, 单克隆抗体, P97蛋白, 表位鉴定

Abstract: In order to prepare a monoclonal antibody (MAb) against Mycoplasma hypneumoniae (Mhp), the splenic cells from BALB/c mice immunized with the P97 C-terminal region of Mhp J strain, which contains R1 region (P97CR1 protein) and was expressed in Escherichia coli (E. coli) BL21(DE3), were fused with SP2/0 myeloma cells, and a hybridoma cell line A3 stably secreting the MAbs against P97 Protein of Mhp were successfully screened. The identification results indicated the A3 MAb was IgG1 subclass with κ light chain. The A3 MAb was able to specifically recognize the P97CR1 protein of Mhp expressed in E. coli BL21(DE3) using Western blot analysis. Flow cytometry analysis showed that the MAb was able to recognize natural P97 protein expressed on the Mhp. The epitope sequence recognized by the A3 MAb was identified as LDDNLQby pepscan technique. The epitope sequence is highly conservative among the P97 proteins of all Mhp strains. The blocking ELISA analysis showed that the binding of the A3 MAb to the P97CR1 protein was blocked by high immune positive serum of Mhp. The preparation of A3 Mab provided foundation for further development of a blocking ELISA assay with high specificity and sensitivity for detecting Mhp infection.

Key words: Mycoplasma hypneumoniae (Mhp), monoclonal antibody (MAb), P97 protein, epitope identification

中图分类号:

王宁, 谨瑾, 刘璐, 刘欢欢, 姜立波, 辛九庆, 刘恒贵. 猪肺炎支原体P97蛋白单克隆抗体的制备、表位鉴定及其初步应用[J]. 畜牧兽医学报, 2020, 51(5): 1110-1118.

WANG Ning, JIN Jin, LIU Lu, LIU Huanhuan, JIANG Libo, XIN Jiuqing, LIU Henggui. Preparation, Identification of the B-cell Epitope and Preliminary Application of a Monoclonal Antibody against P97 Protein of Mycoplasma hypneumoniae[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 1110-1118.

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