| 小鼠PD-1胞外段的克隆、原核表达及活性分析 |
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| 引用本文： | 覃晓琳,刘朝奇,李斌,吕佰瑞,杨凡. 小鼠PD-1胞外段的克隆、原核表达及活性分析[J]. 免疫学杂志, 2010, 26(5) |
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| 作者姓名： | 覃晓琳 刘朝奇 李斌 吕佰瑞 杨凡 |
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| 作者单位： | 三峡大学分子生物学研究所,宜昌,443002;三峡大学分子生物学研究所,宜昌,443002;三峡大学分子生物学研究所,宜昌,443002;三峡大学分子生物学研究所,宜昌,443002;三峡大学分子生物学研究所,宜昌,443002 |
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| 基金项目： | 三峡大学研究生科研创新基金项目(2009-36) |
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| 摘 要： | 目的小鼠PD-1胞外段(ePD-1)原核表达载体的构建、表达、纯化及其生物学效应初步研究。方法应用小鼠脾细胞总RNA,采用RT-PCR克隆PD-1胞外段基因,将其重组到原核表达载体pGEX-4T-1中,构建原核表达载体pGEX-4T-1-ePD-1,转化至E.coli DH5α,筛选阳性菌落,进行酶切及测序鉴定。将测序正确的重组表达质粒pGEX-4T-1-ePD-1转化至E.coli BL21(DE3)中,经IPTG诱导表达后,对表达产物进行SDS-PAGE和Western blot检测,同时通过蛋白质纯化仪纯化GST-ePD-1融合蛋白。利用流式细胞术和Alamar Blue法,检测纯化的PD-1胞外段融合蛋白对淋巴细胞增殖的影响,评价其生物学活性。结果成功构建重组质粒pGEX-4T-1-ePD-1,并在E.coli BL21(DE3)中获得了成功表达,利用蛋白质纯化仪得到纯化的GST-ePD-1融合蛋白。流式细胞术和Alamar Blue法结果均显示,不同浓度的融合蛋白作用于混合淋巴细胞,与阴性对照相比,可明显促进淋巴细胞的增殖。结论成功地克隆、表达和纯化了小鼠ePD-1蛋白,纯化的重组蛋白可有效促进淋巴...
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| 关 键 词： | 小鼠PD-1胞外段 融合蛋白 蛋白纯化 淋巴细胞增殖 |
Cloning and expression of the mouse sPD-1 as well as its biological activity analysis |
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| QIN Xiaolin,LIU Chaoqi,LI Bin,LV Bairui,YANG Fan Institute of Molecular Biology,Three Gorges University,Yichang ,China. Cloning and expression of the mouse sPD-1 as well as its biological activity analysis[J]. Immunological Journal, 2010, 26(5) |
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| Authors: | QIN Xiaolin LIU Chaoqi LI Bin LV Bairui YANG Fan Institute of Molecular Biology Three Gorges University Yichang China |
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| Affiliation: | QIN Xiaolin,LIU Chaoqi,LI Bin,LV Bairui,YANG Fan Institute of Molecular Biology,Three Gorges University,Yichang 443002,China |
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| Abstract: | To investigate the biological activity of mouse PD-1 ectodomain(ePD-1),we cloned and expressed ePD-1 gene. ePD-1 gene was amplified from mouse spleen cell mRNA by RT-PCR,and then cloned into prokaryotic expression vector pGEX-4T- 1,for screening positive recombinant plasmid pGEX-4T-1-ePD-1 by enzyme digestion and sequencing.Then the correct sequence of the recombinant plasmid was transformed into E.coli BL21,and induced by IPTG.The expressed recombinant protein was analyzed by SDS-PAGE and Western blotting;... |
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| Keywords: | Mouse ePD-1 Fusion protein Protein purification Lymphocyte proliferation |
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