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目的:制备高效价的特异性小鼠Foxp3抗血清,并初步应用于正常小鼠组织的Foxp3表达的鉴定。方法:构建重组质粒pGEX-6p-2/Foxp3,并转化至E.coliBL21(DE3)中进行大量诱导表达,用纯化的重组融合蛋白免疫新西兰兔,制备抗血清。以制备的抗血清为一抗,Western blot检测小鼠组织的Foxp3蛋白的表达。结果:SDS-PAGE及Western blot鉴定,诱导表达及纯化的重组融合蛋白能被抗GST单克隆抗体(mAb)识别,在Mr45000附近有特异条带,与预期融合蛋白大小一致,获得的抗血清效价在1∶12800以上,该抗血清能特异性识别NIH3T3细胞中表达的Foxp3蛋白。Westernblot鉴定结果显示在脾脏、胸腺、淋巴结中Foxp3蛋白有较高表达,胃也有少量的表达,而骨骼肌、脂肪组织未见表达。结论:制备了高效价的特异性小鼠Foxp3抗血清,并可用于正常小鼠组织的Foxp3蛋白的表达鉴定,为进一步研究Foxp3奠定了实验基础。
OBJECTIVE: To prepare high-titer specific mouse anti-Foxp3 antiserum and identify the expression of Foxp3 in normal mouse tissues. Methods: The recombinant plasmid pGEX-6p-2 / Foxp3 was constructed and transformed into E. coli BL21 (DE3) for inducing in large quantities. The purified recombinant fusion protein was used to immunize New Zealand rabbits to prepare antiserum. The prepared antiserum was used as the primary antibody, and the expression of Foxp3 protein was detected by Western blot. Results: The fusion protein induced by anti-GST monoclonal antibody (mAb) was identified by SDS-PAGE and Western blot. The specific fusion protein was found in the vicinity of Mr45000, which was consistent with the size of the expected fusion protein. The antiserum efficacy At a price above 1: 12800, the antiserum specifically recognizes Foxp3 protein expressed in NIH3T3 cells. Western blot showed that Foxp3 protein was expressed in the spleen, thymus and lymph nodes in a small amount, but not in skeletal muscle and adipose tissue. CONCLUSION: High titer specific mouse anti-Foxp3 antiserum was prepared and used to identify the expression of Foxp3 protein in normal mouse tissues, which laid the foundation for further study of Foxp3.