TeiR融合蛋白分离及多克隆抗体的制备  

Separation of TeiR fusion protein and its multiclonal antibody preparation

作　　者：陈建秋[1] 艾育芳[2] 潘大仁[2] 周以飞[1] 潘菲[2] 林彬辉[2] 郭玉春[1] 

机构地区：[1]福建农林大学作物科学学院 [2]福建农林大学生命科学学院,福建福州350002

出　　处：《福建农林大学学报（自然科学版）》2009年第3期255-259,共5页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：教育部科学技术重点项目(207056);福建省自然科学基金资助项目(2008J0041);福建省教育厅科学技术重点项目(JA06011);福建省发改委资助项目(闽计投资[2003]170号)

摘　　要：提取睾丸酮丛毛单胞菌(Comamonas testosteroni)基因组DNA,PCR扩增teiR基因,将扩增产物克隆到pET28a中,经酶切验证后获得teiR基因表达载体pET28a-teiR.将重组质粒转化大肠杆菌(Escherichia coli)BL21,添加终浓度为1 mmol.L-1的IPTG进行诱导,提取细菌总蛋白进行蛋白的SDS-PAGE电泳.并根据融合蛋白特性,利用Ni-NTA Spin Column对TeiR融合蛋白进行分离纯化,经分离纯化后的重组蛋白在SDS-PAGE上呈现出单一条带.进一步以获得的TeiR融合蛋白为抗原,制备兔抗TeiR多克隆抗体,经ELISA测定该抗体的效价为1∶10000,该抗体与睾丸酮丛毛单胞菌天然TeiR蛋白反应良好,说明该抗体特异性良好.The teiR gene was amplified by PCR from the genomic DNA of Comamonas testosteroni and then cloned into plasmid pET28a to construct recombinant plasmid pET28a-teiR. Plasmid pET28a-teiR was transformed into Escherichia coli BL21. The total cell protein of the host bacteria was extracted for SDS-PAGE after inducing with 0.001 mol · L-1 IPTG. TeiR fusion protein was pu- rified using Ni-NTA Spin Column for muhiclonal antibody preparation. The results of ELISA showed that the titer of the muhiclonal antibody against the purified TeiR fusion protein was about 1： 10000. The antibody reacted well against the native TeiR protein from C. testosteroni.

关 键 词：睾丸酮丛毛单胞菌 teiR SDS—PAGE 多克隆抗体 

分 类 号：X172[环境科学与工程—环境科学]