小鼠Foxp3抗血清的制备及应用_临床医学论文.docVIP

小鼠Foxp3抗血清的制备及应用_临床医学论文 小鼠Foxp3抗血清的制备及应用_临床医学论文 作者：焦海春, 肖建华*, 何涛君, 胡杰, 崔彦芬 【摘要】 　　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并转化至E.coli BL21(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Mr 45000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。Western blot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。 【关键词】 Foxp3 CD4+CD25+Treg细胞 抗血清制备 　　[Abstract] AIM: To prepare the high titer and specific antihain reactions(PCR) and cloned into the prokaryotic expression vector pGEX6p2. The recombinant plasmid pGEX6p2/Foxp3 was transformed into E.coli BL21(DE3) to be expressed. The expressed fusion protein GSTFoxp3 was analyzed by SDSPAGE and Western blot. The polyclonal clantiserum was obtained by immunizing New Zealand rabbits with the purified fusion protein as antigen. The Foxp3 expression in normal mouse tissues was detected by Western blot with the antiserum. RESULTS: The expressed products were analyzed by SDSPAGE and Western blot. The results showed that the molecular weight of the expressed protein was about 45 000. The titer of the antiserum was above 1∶12 800. We observed that the antiserum could recognize Foxp3 protein expressed in NIH3T3 cells by immunoblotting. Western blot results showed that the Foxp3 protein was expressed highly in spleen, thymus and lymph nodes, less expressed in stomach, but not expressed in skeletal muscle and adipose tissue. CONCLUSION: High titer antiserum against mouse Foxp3 is produced and can be used to identify the Foxp3 expression in normal mouse tissues. 　　[Keywords]Foxp3; CD4+CD25+Treg cell; antiserum preparation 　　1995年, Sakaguchi等[1]发现CD4+CD25+调节性T淋巴细胞（regulatory T cell, Treg）后, 该细胞的研究成了近年来免疫学领域的研究热点之一。人们发现, Treg在自身免疫病、 抗感染免疫、 肿瘤免疫、 移植物耐受等方面都具有重要意义。Foxp3基因属于转录因子forkhead/winged家族, 该基因家族由大量分布极广的转录因子组成, 该家族成员