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当前位置: 首页 > 文章 > 山羊SOX2多克隆抗体制备 中国农业科学 2012,45 (1) 178-183
Position: Home > Articles > Preparation of Polyclonal Anti-Sox2 Antibody in Capra hircus Scientia Agricultura Sinica 2012,45 (1) 178-183

山羊SOX2多克隆抗体制备

作  者：
刘平;张昀;郑喜邦;李恭贺;岑小妹;岳磊磊;宗自杰;卢晟盛;卢克焕;张明
单  位：
广西大学动物科学与技术学院
关键词：
Sox2;原核表达;蛋白纯化;多克隆抗体;山羊
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。
译  名：
Preparation of Polyclonal Anti-Sox2 Antibody in Capra hircus
作  者：
LIU Ping,ZHANG Yun,ZHENG Xi-bang,LI Gong-he,CEN Xiao-mei,YUE Lei-lei,ZONG Zi-jie,LU Sheng-sheng,LU Ke-huan,ZHANG Ming(College of Animal Science and Technology,Guangxi University,Nanning 530005)
关键词：
Sox2 gene;prokaryotic expression;protein purification;polyclonal anti body;Capra Hircus
摘  要：
【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).

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