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LPTS抗体的制备和活性检测
引用本文:李义良,宋海,赵静,张红锋,李载平,赵慕钧.LPTS抗体的制备和活性检测[J].中国生物工程杂志,2004,24(3):44-47.
作者姓名:李义良  宋海  赵静  张红锋  李载平  赵慕钧
作者单位:1. 中国科学院上海生物化学与细胞生物学研究所, 上海 200031; 2. 华东师范大学生命科学学院, 上海 200062
基金项目:国家“8 63”计划资助项目 (2 0 0 2BA711A0 2 )
摘    要:LPTS是利用定位候选克隆策略 ,得到的一个新的肝相关候选肿瘤抑制基因 (anovelliver relatedputativetumorsuppressor) ,为了进一步研究其结构与功能 ,利用DNA重组技术 ,将LPTS的cDNA克隆到融合表达载体pET 2 4a中 ,在E .coli中表达 ,以Ni+柱亲和层析 ,获得纯化的 6×His LPTS融合蛋白。以此为抗原免疫新西兰大白兔获得多克隆抗体 ,ELISA法检测其滴度达 2 0 0 0 0以上 ,经亲和层析纯化 ,Western印迹结果表明 ,该纯化抗体可与真核表达的HA LPTS蛋白和内源性的LPTS蛋白特异性结合 ;免疫荧光分析显示SMMC 772 1细胞内源性表达的LPTS蛋白呈点状分布于细胞核内。以上结果表明获得了效价高 ,活性强的针对LPTS蛋白的多克隆抗体 ,可用于对LPTS的结构和功能研究。

关 键 词:LPTS  多克隆抗体  亲和层析  免疫荧光  特异性  
收稿时间:2003-12-08
修稿时间:2003年12月8日

Preparation and Identification of Polyclonal Antibodies Against LPTS
LI Yi liang , SONG Hai ZHAO Jing ZHANG Hong feng LI Zai ping ZHAO Mu jun.Preparation and Identification of Polyclonal Antibodies Against LPTS[J].China Biotechnology,2004,24(3):44-47.
Authors:LI Yi liang  SONG Hai ZHAO Jing ZHANG Hong feng LI Zai ping ZHAO Mu jun
Affiliation:LI Yi liang 1,2 SONG Hai 1 ZHAO Jing 1 ZHANG Hong feng 2 LI Zai ping 1 ZHAO Mu jun 1
Abstract:LPTS encodes a novel liver relatived putative tumor suppressor,which was isolated by allelic loss mapping and positional candidate cloning.In order to study the structre and function of LPTS, the coding region of LPTS gene was successfully sub cloned into the prokaryotic expression vector pET 24a.The LPTS expression plasmid was then introduced into E.coli BL21(DE3) cells for highly efficient expression.His LPTS fusion protein was purified by affinite chromatography,then this protein was used to immunize rabbits to produce polyclonal antibodies.The polyclonal antibodies was also purified by affinite chromatography. The specificity of the anti LPTS antibody was successfully proved by western blotting analysis on eukaryotic expressed products of LPTS and HA LPTS protein.Moreover,LPTS protein was found to be distributed only in nuclear of SMMC?7721 cell line by immunofluorescence assay using anti LPTS antibodies.All of the results demonstrated that the polyclonal antibodies for LPTS could be further used for analyzing the structure and function of LPTS protein.
Keywords:LPTS  Polyclonal antibodies Affinite chromatography Specificity immunofluorescence
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