山羊SOX2多克隆抗体制备

【目的】构建山羊 Sox2 原核表达载体-pRSET-Sox2，并将诱导表达、纯化的 His-Sox2 融合蛋白免疫新西兰大白兔，制备 Sox2 多克隆抗体。【方法】从 pMD18T-Sox2 载体上以 Bam H I和 Xho I 双酶切截取 Sox2 片段，然后将其亚克隆到 pRSET-A 表达载体上，获得 pRSET-Sox2 重组质粒。转化了 pRSET-Sox2 的大肠杆菌 BL21(DE3)，1 mmol?L-1 IPTG  37℃ 诱导 4 h，SDS-PAGE电泳及 Western blotting 检测融合蛋白表达。相同条件下大量增菌诱导，用Ni- NTA argrose 介质分离纯化 His-Sox2 重组蛋白。将体外复性的融合蛋白皮下注射新西兰大白兔，间隔 2-3 周注射一次，共 4 次。最后一次注射后 7 d，采血分离血清，用 Western blotting 检测抗体特异性。【结果】（1）原核表达载体 pRSET-Sox2 在大肠杆菌 BL21(DE3) 得到了高效表达；（2）纯化的 His-Sox2 融合蛋白能够满足多克隆抗体制备的要求；(3)经 Western blotting 检测，Sox2 多克隆抗体能与 His-Sox2 融合蛋白特异性结合。【结论】制备了高特异性山羊 Sox2 多克隆抗体，为深入探讨山羊 Sox2 基因的生物学功能奠定了基础，也为山羊（iPS）细胞检测创造了良好条件。

Preparation of Polyclonal Anti-Sox2 Antibody in Capra hircus

【Objective】 The present study was to construct a prokaryotic expression vector of Capra hircus Sox2 gene, pRSET-Sox2, to induce expression and purification of His-Sox2 fusion protein, which was used to immunize New Zeland white rabbits to prepare polyclonal anti-Sox2 antibody. 【Method】 Removed from plasmid pMD18-Sox2 by double digestion of BamH I and Xho I, Sox2 fragment was subcloned to pRSET-A vector to construct recombinant plasmid pRSET-Sox2. The plasmid was transformed into E. coli BL 21 (DE3), and His-Sox2 fusion protein was induced to expess with 1 mmol?L-1 IPTG at 37℃ for 4 h, which was identified with SDS-PAGE analysis and Western blotting. In the same way, large volume of expressing culture was prepared to purify His-Sox2 fusion protein with NI-NTA argrose under denaturing condition. The refolded fusion protein in vitro was injected subcutaneously into New Zeland white rabbits for four times at intervals of 2-3 weeks. Seven days after the last injection, blood samples were collected, serum was isolated, and specificity of polyclonal anti-Sox2 antibody was determined by Western blotting assay.【Result】 The prokaryotic expression vector pRSET-Sox2 was expressed efficiently in E. coli. BL21. The purified His-Sox2 was qualified for preparation of polyclonal antibody. The polyclonal anti-Sox2 antibody was prepared, and it could bind His-Sox2 fusion protein specifically, which was illustrated by Western blotting assay. 【Conclusion】 The polyclonal anti-Sox2 antibody with strong specificity was prepared, which will lay a solid biological foundation for study of Sox2, and for its application in detection of Capra hircus iPS cells (induced pluripotent stem cells).