摘要
【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。
[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 pMD 18-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）.
出处
《中国农业科学》
CAS
CSCD
北大核心
2012年第1期178-183,共6页
Scientia Agricultura Sinica
基金
广西自然科学基金项目(桂科自0728019
桂科自0991043)
广西亚热带生物资源保护与利用重点实验室开放课题(SB0907)
关键词
SOX2
原核表达
蛋白纯化
多克隆抗体
山羊
Sox2 gene
prokaryotic expression
protein purification
polyclonal anti body
Capra Hircus
