泉古菌Sulfolobus islandicus PCNA1的原核表达及其多克隆抗体的制备  

Prokaryotic Expression of PCNA1 from Crenarchaeaon Sulfolobus islandicus and Preparation of Its Polyclonal Antibody

作　　者：田斌[1] 张昌毅 佘群新[1] 梁运祥[1] 

机构地区：[1]华中农业大学生命科学技术学院/农业微生物学国家重点实验室,武汉430070

出　　处：《华中农业大学学报》2010年第1期59-62,共4页Journal of Huazhong Agricultural University

基　　金：科技部"973"计划项目(2003CB716004)资助

摘　　要：泉古菌Sulfolobus islandicus中PCNA有3个同源类似蛋白,本研究选取PCNA1作为研究对象,为获得可溶性表达的PCNA1蛋白,制备多克隆抗体,深入了解PCNA1基因的功能,将PCNA1基因片段克隆到组氨酸标签融合的表达载体pET30a中,利用IPTG诱导,金属螯合亲和层析进行纯化分析表明,融合蛋白大部分可溶。紫外分光光度法测定纯化蛋白的纯度可达90%以上,浓度约为2 mg/mL。免疫家兔制备多克隆抗体,ELISA检测抗血清效价可达1∶10 000以上,Western印迹检测证明抗体特异性良好。Based on the result of sequence analysis,there are three homologues of PCNA in Crenarchaeaon Sulfolobus islandicus and PCNA1 was selected as the subject of the current study. To obtain soluble protein of PCNA1 and its polyclonal antibody for studying the function of PCNA1 gene,the fragment of PCNA1 gene was amplified and cloned into the expression vector pET30a. Induced by IPTG, soluble protein of interest could be obtained. The protein was purified by metal chelate affinity chroma- tography and its purity and content were detected with UV-spectrophotometric method. Rabbits were immunolized by PCNA1 protein. The titer and specificity of polyclonal antibody were detected with indirect ELISA assay and Western blotting. The result of UV-spectrophotometric method showed that its purity reached at least 90% and the protein concentration was about 2 mg/mL. The titer of antiserum was as high as 1 ： 10 000,with a very high specificity detected by the Western blotting.

关 键 词：泉古菌 PCNA1基因 原核表达 金属螯合亲和层析 多克隆抗体 

分 类 号：Q939.9[生物学—微生物学]