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 。
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.
出处
《中国生物工程杂志》
CAS
CSCD
2004年第3期44-47,共4页
China Biotechnology
基金
国家"8 63"计划资助项目 (2 0 0 2BA711A0 2 )
关键词
LPTS
抗体
活性
亲和层析
免疫荧光
特异性
肝癌
LPTS Polyclonal antibodies Affinite chromatography Specificity immunofluorescence