粉尘螨变应原ProDer f 1编码基因重组pET28a-YARA-Ihc体系的构建及表达 |
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引用本文: | 刘志明,;姜玉新.粉尘螨变应原ProDer f 1编码基因重组pET28a-YARA-Ihc体系的构建及表达[J].热带病与寄生虫学,2014(3):125-128. |
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作者姓名: | 刘志明 ;姜玉新 |
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作者单位: | [1]东南大学医学院附属南京江北人民医院,南京市 210048; [2]皖南医学院寄生虫学教研室,南京市 210048; |
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基金项目: | 国家自然科学基金资助项目(81172790) |
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摘 要: | 目的构建粉尘螨变应原ProDerf1原核表达载体pET28a-YARA—IhC—ProDerf1,为评价其免疫治疗效果奠定基础。方法用分子克隆技术构建出表达载体pET28a—YARA—IhC—ProDerf1,在E.coli BL21(DE3)中表达融合蛋白YARA-IhC-ProDer f 1,并进行Ni~(2+)-NTA树脂柱亲和层析以纯化蛋白。结果经测序证实成功构建了表达载体pET28a-YARA-IhC-ProDer f 1,YARA-IhC-ProDer f 1融合蛋白在E.coli BL21(DE3)中得到表达,纯化后的蛋白浓度为278μg/ml。SDS-PAGE和Westem blot分析表明纯化蛋白为目的蛋白YARA-IhC-ProDer f 1。结论已成功制备出pET28a—YARA—IhC-ProD—er f 1原核表达载体,融合蛋白得到表达和纯化。
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关 键 词: | 细胞穿透肽 粉尘螨 恒定链 变应原 |
Construction and expression of the recombinant plasmid pET28a-YARA-Ihc-ProDer f 1 of Derma-tophagoides farina |
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Institution: | Liu Zhiming, Jiang Yuxin(1. Department of clinical laboratory, Affiliated Nanjing fiangbei Hospital of Medical College, Southeast University Medical College, Nanjing 210048, China. 2. Department of Medical Parasitology, Wannan Medical College.) |
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Abstract: | Objective To construct the prokaryotic expression vector pET28a-YARA-Ihc-ProDer f 1 and provide the foundation for exploring the fusion protein effect as vaccine for specific immunotherapy. Methods Two oligonucleotides encoding YARA were synthesized and annealed to generate YARA-encoding DNA. The fused genes, YARA-Ihc-ProDer f 1 was constructed and inserted into the prokaryotic expression vector pET28a(+). The fusion protein YARA-Ihc-ProDer f 1 induced with IPTG in E.coli BL21(DE3) was purified with Ni2+-resin affinity chromatography and confirmed with SDS-PAGE and Western blot. Results Sequence analysis confirmed the construction of the expression vector pEt28a-YARA-Ihc-ProDer f 1 successfully, the fusion protein YARA-Ihc-ProDer f 1 was expressed and purified with the concentration of 278μg/ml. SDS-PAGE and Western blot demonstrated the fusion protein was YARA-Ihc-ProDer f 1. Conclusion The recombinant prokaryotic expression vectors, pET28a(+)-YARA-Ihc-ProDer f 1 was successfully constructed, and the fusion protein was expressed and purified . |
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Keywords: | Cell penetrating peptides Dermatophagoidesfarina invariant chain allergens |
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