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PA28γ蛋白原核表达纯化及其多克隆抗体制备 被引量：1

Construction of Recombinant PA28γ Plasmid and Expression and Preparation of Polyclonal Antiserum
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摘要 目的蛋白酶体激活亚单位3(PA28γ)的表达、纯化及其多克隆抗体的制备。方法利用pGEX-4T-1原核表达系统表达PA28γ蛋白,经T-A克隆、亚克隆至pGEX-4T-1表达载体;将重组质粒转化到大肠杆菌BL-21-STAR(DE3),诱导表达PA28γ蛋白,通过Glutathione Sepharose 4B亲和层析纯化融合蛋白(GST-PA28γ);并用其免疫BALB/c小鼠制备多抗血清,对表达产物和多抗血清进行Western-blot鉴定。结果成功构建原核表达质粒PA28γ/pGEX-4T-1,且测序正确;获得高纯度的PA28γ蛋白及其高效价的多克隆抗体血清,并得到Western-blot证实。结论成功利用基因重组技术制备了PA28γ蛋白和抗PA28γ多克隆血清,为该蛋白的生物学功能研究奠定了基础。 Objective To express PA28γ antigen as a GST- PA28γ fusion protein and prepared anti-PA28γ antiserum. Methods The PA28γgene was cloned into the vector pGEX-4T-1. The recombinant plasmid was transferred into E.coli BL-21-STAR （DE3） and the GST- PA28γfusion protein was expressed by inducing with IPTG. The fusion protein was purified by glutathione sepharose 4B affinity chromatography. BALB/c mice were immunized with recombinant PA28γ, and western blot was used to detect the expressed protein and polyclonal antiserum. Results DNA sequencing confirmed the PA28γsequence was correct. Expressed fusion protein was mainly in soluble supernatant and accounted for about 15% of the total bacterial proteins. Western blot results demonstrated the recombinant GST- PA28γ was antigenic and could induce antibody production in mice. Conclusion The successful preparation of PA28γprotein and anti-PA28γpolyclonal antiserum facilitates further studies on the structure, functions and protein-protein interaction of PA28γ.
作者 赵玉梅 王穗海 潘华政 黄湘 李明
机构地区 南方医科大学生物技术学院
出处 《热带医学杂志》 CAS 2008年第3期201-204,共4页 Journal of Tropical Medicine
基金 国家863计划项目(No.2006AA02A311)
关键词 PA28γ 原核表达 纯化 多克隆血清 PA28γ expression purification polyclonal antiserum
分类号 R73-362 [医药卫生—肿瘤]
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同被引文献17

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热带医学杂志
2008年 第3期

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