山羊附睾头上皮细胞gBD124互作蛋白的筛选与鉴定

Screening and identification of gBD124-interacting proteins in goat epididymal head epithelial cells

摘要:

本研究旨在筛选gBD124的互作蛋白，为其在山羊育种繁殖中的功能研究提供依据。以纯化后的重组gBD124蛋白免疫小鼠，采用杂交瘤技术融合脾细胞与骨髓瘤细胞，筛选获得特异性单抗细胞株。取效价为1.12×10⁵的gBD124单抗进行免疫共沉淀联合质谱分析，初筛得到候选的互作蛋白；继而利用山羊附睾头上皮细胞进行免疫荧光共定位进一步筛选，并再次通过免疫共沉淀(co-immunoprecipitation, Co-IP)验证。结果表明，共获得了5株细胞株，其效价介于0.89×10⁴-1.12×10⁵之间，选择效价最高(1.12×10⁵)的细胞株，制备获得gBD124单克隆抗体。应用Co-IP联合质谱技术，从山羊附睾头组织蛋白中鉴定出2个潜在的gBD124互作蛋白，即核纤层蛋白A/C (lamin A/C, LMNA)和热休克蛋白90α家族A类成员1 (heat shock protein 90 alpha family class A member 1, HSP90AA1)；免疫荧光共定位结果显示gBD124与HSP90AA1共存于细胞质，而gBD124与LMNA则共定位于细胞核。当使用gBD124抗体进行固定后，Co-IP复合物中含有HSP90AA1却不含LMNA，从而证实HSP90AA1是与gBD124发生相互作用的蛋白。对山羊附睾组织进行荧光定量和Western blotting表达分析后发现，gBD124在附睾头1的表达量较高，显著高于附睾其他组织(P0.05)；同时，HSP90AA1在附睾头中的表达量也处于较高水平(P0.05)。本研究获得了gBD124单克隆抗体，筛选并鉴定了gBD124的互作蛋白HSP90AA1，为阐明β防御素124在雄性生殖系统中的新型互作蛋白及其潜在信号网络提供了参考。

Abstract:

This study used the prepared gBD124 monoclonal antibody to explore the interacting proteins of gBD124 through co-immunoprecipitation (Co-IP) combined with mass spectrometry, aiming to provide a basis for further exploration of the role of gBD124 in goat breeding and reproduction. Mice were immunized with the purified recombinant gBD124 protein, and specific monoclonal antibody-producing cell lines were obtained by fusing spleen cells and myeloma cells through the mouse monoclonal hybridoma technology. The prepared gBD124 monoclonal antibody with a titer of 1.12×10⁵ was used for Co-IP experiments, and the possible target proteins were screened by mass spectrometry. Then, the goat epididymal head epithelial cells were used for immunofluorescence co-localization to screen the proteins interacting with gBD124, and the results were further verified by Co-IP. The results showed that five cell lines with titers ranging from 0.89×10⁴ to 1.12×10⁵ were obtained, and the gBD124 monoclonal antibody with the highest titer of 1.12×10⁵ was used in the subsequent experiments. Two possible proteins, lamin A/C (LMNA) and heat shock protein 90α family class A member 1 (HSP90AA1), interacting with gBD124 were screened from the goat epididymal head tissue proteins by Co-IP combined with mass spectrometry. The immunofluorescence co-localization results showed that gBD124 and HSP90AA1 were co-localized in the cytoplasm, while gBD124 and LMNA were co-localized in the nucleus. The Co-IP complex fixed by gBD124 antibody contained HSP90AA1 but no LMNA, indicating that HSP90AA1 was the interacting protein of gBD124. The expression analysis of gBD124 and HSP90AA1 in the goat epididymal tissue by fluorescence quantitative PCR and Western blotting showed that the expression of gBD124 was higher in the epididymal head 1 than in other epididymal tissue (P0.05). HSP90AA1 also exhibited higher expression in the epididymal head (P0.05). This study obtained the gBD124 monoclonal antibody and identified the interacting protein HSP90AA1 of gBD124, providing a reference for clarifying the novel interacting proteins and potential signaling networks of β-defensin 124 in the male reproductive system.