抗mEET蛋白单克隆抗体的制备、鉴定及mEET组织学分布的分析

Preparation and characterization of monoclonal antibodies against mouse estrogen-enhanced transcript

目的 制备抗小鼠雌激素增强的转录子（mouse estrogen-enhanced transcript，mEET）的单克隆抗体（monoclonal antibody，McAb），探讨mEET蛋白在小鼠各组织器官中的定位与分布，为进一步了解和研究mEET功能奠定基础。方法 以纯化的原核表达mEET蛋白免疫的SD大鼠脾细胞与Sp2／0融合，经HAT筛选获得稳定分泌抗mEET蛋白的McAb的杂交瘤。采用McAb通过免疫组织化学及间接免疫荧光染色分析mEET在胸腺上皮细胞系MTEC1及各组织器官中的定位与分布。结果 获得4株能高效分泌特异性McAb的抗mEET的杂交瘤细胞系DA10、DB4、4A5和4D8，Ig亚类均为IgM，其腹水效价为1：10^7～1：10^8。Western blot结果表明该抗体能特异性识别原核表达的mEET蛋白，免疫荧光及免疫组化显示mEET蛋白在胞浆点状弥散分布，部分细胞可见核内分布。免疫组化提示该蛋白在全身多个器官有表达，尤其在骨髓中的巨核细胞胞浆中高表达。结论 成功获得抗mEET的特异性的单克隆抗体，为进一步研究mEET的功能及在胸腺发育中的作用创造了条件。

Objective In order to explore the tissue and sub-cellular distribution of mouse estrogen-enhanced transcript（mEET） protein, we prepared and characterized monoclonal antibodies against mEET. Methods Hybridomas were generated by fusion of Sp2/0 myelomas and spleen cells from rats immunized with mEET recombinant proteins. Hypoxanthine-aminopterin-thymidine-resistant clones were then screened for secretion of antibodies specific for mEET by ELISA and identificated by Western blot. The monoclonal antibodies were subsequently used for studies of tissue distribution and sub-cellular localization of mEET protein by immunofluorescence or immunohistochemistry. Results Four bybridoma cell lines, DA10, DB4, 4A5 and 4138, were produced, each of which stably secrets antibodies again mEET. Isotyping indicated that they were IgM antibodies. Immunofluorescent assay demonstrated that mEET protein was mainly distributed in cytoplasm, whereas immunochemistry study showed wide tissue distribution with particularly high levels of expression in megakaryocytes in bone marrow. Conclusion The monoclonal antibodies against mEET protein have been successfully prepared, which should provide useful reagent for further investigation into the biological function of mEET.