Abstract: To elucidate the biological functions of GAM56 protein in Eimeria tenella，the coding sequence of the gam56 gene was amplified from the cDNA of gametocytes isolated from the Guangdong strain of E. tenella. Subsequently，a truncated prokaryotic expression vector pET⁃B2M⁃GAM56 was constructed，and the recombinant GAM56 protein was expressed in E. coli expression system using IPTG induction. Polyclonal antibodies were generated in BALB/c mice immunized with purified GAM56. The antibody titer and specificity were analyzed using indirect ELISA and Western blot. Gametocyte⁃stage samples of E. tenella（GD）were collected for immunofluorescence assay to evaluate antibody performance. Results revealed that compared with the reference E. tenella（Houghton）strain，the gam56 gene of E. tenella（GD）exhibited a partial terminal deletion，presenting a 1 077 bp CDS encoding 358 amino acids （predicted molecular weight 41.3 kDa）. The truncated GAM56 protein containing conserved regions was predominantly expressed in inclusion bodies. Indirect immunofluorescence assay demonstrated specific recognition of the GAM56 protein at the gametocyte stage，with subcellular localization consistent with literature reports. This study provides critical materials for elucidating the biological functions of GAM56 in the E. tenella（GD）and establishes a foundation for functional investigations of sexual development⁃associated proteins in Eimeria species.

Key words: Eimeria tenella, GAM56, Prokaryotic expression, Polyclonal antibodies

摘要： 为研究柔嫩艾美耳球虫GAM56蛋白的生物学功能，以柔嫩艾美耳球虫广东株配子 体cDNA为模板，克隆该分离虫株的gam56 基因编码序列。构建pET?B2M?GAM56截短表达载 体，利用大肠杆菌表达系统通过IPTG诱导表达GAM56蛋白，纯化后免疫BALB/c小鼠制备 GAM56多克隆抗体。通过间接ELISA和Western blot分析抗体的效价和特异性；收集E. tenella （GD）配子体阶段样品，利用GAM56多克隆抗体进行免疫荧光检测以评价其应用效果。结果显 示：相比E. tenella（Houghton）参考虫株，E. tenella（GD）的gam56 基因存在末端部分片段的缺 失，CDS序列1 077 bp，可编码358个氨基酸；预测蛋白大小41.3 kDa。GAM56保守区域截短原 核表达蛋白主要以包涵体形式存在。间接免疫荧光分析显示该抗体能够特异性识别配子体阶 段的GAM56蛋白。该研究为进一步解析柔嫩艾美耳球虫广东株GAM56生物学功能提供了材 料，为艾美耳球虫有性发育相关蛋白的功能研究与机制解析奠定基础。

关键词: 柔嫩艾美耳球虫, GAM56, 原核表达, 多克隆抗体