| 摘要: |
| 采用 PCR 方法扩增团头鲂组成型 HSC70 和诱导型 HSP70 基因完整的编码区片段, 并分别克隆到原核表达载体 pET-22b(+)中, 然后转化大肠杆菌 BL21(DE3), 用 1mmol/L IPTG 在不同温度及时间下进行诱导表达。 采用 Ni-NTA His Bind Resins 亲和层析和 DEAE-Sepharose FF 阴离子交换柱层析对目的蛋白进行纯化, 并进行 SDS-PAGE 和 Western-blotting 分析。结果表明, 成功构建了团头鲂两种重组表达质粒 pET-22b(+)/Ma-HSC70 和 pET-22b(+)/Ma-HSP70, 表达融合蛋白的相对分子量均约为72kDa, 并能与兔抗人HSP70多抗进行特异性结合, 这两种HSP70s融合蛋白经纯化后的纯度均达到 95%以上。本实验选择融合蛋白 Ma-HSC70 在 25℃和 Ma-HSP70 在 30℃下分别诱导 7h 作为可溶性表达的最佳条件。 |
| 关键词: 团头鲂, 热休克蛋白 70, 原核表达, 融合蛋白, 纯化, 鉴定 |
| DOI：10.11693/hyhz201201030030 |
| 分类号: |
| 基金项目:农业部行业专项, 200803013 号; 现代农业产业技术体系建设专项, nycytx-49-18 号 |
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| PROKARYOTIC EXPRESSION, PURIFICATION AND IDENTIFICATION OF TWO HSP70s IN WUCHANG BREAM MEGALOBRAMA AMBLYCEPHALA |
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MING Jian-Hua1,2,3, XIE Jun2, XU Pao2, GE Xian-Ping2, LIU Wen-Bin4, YE Jin-Yun1
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1.College of Life Sciences, Huzhou Teachers College;2.Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences;3.Wuxi Fisheries College,;4.Wuxi Fisheries College, Nanjing Agricultural University
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| Abstract: |
| In this research, The fragments of Constitutive HSC70 and inducible HSP70 ORFs of Wuchang bream Megalobrama amblycephala were amplified by PCR, and then cloned into prokaryotic expression vector pET-22b(+). The two recombinant plasmids, confirmed by double-endonuclease digestion and DNA sequencing, were transformed into competent E. coli BL21 (DE3). The two recombinant strains were induced by 1mmol/L IPTG at different temperatures and times, establishing optimal conditions for inducible expression. The expression products were purified by Ni-NTA His Bind Resins affinity chromatography and DEAE-Sepharose FF anion-exchange chromatography, and detected by SDS-PAGE and Western-blotting. The results show that two recombinant expression plasmids of pET-22b(+)/Ma-HSC70 and pET-22b(+)/Ma-HSP70, each expressing a 72kDa protein that could be recognized by rabbit source anti-HSP70 polyclonal antibody, were successfully constructed, and the purity of two purified fusion proteins was more than 95%. A higher temperature (37℃) was conducive to the rapid expression of fusion protein, which was easy to form inclusion body; a lower temperature (25℃) was in favor of the soluble expression of fusion protein, but also reduced the expression rate of fusion protein. All things considered, the optimal conditions for expressions of two fusion proteins of Ma-HSC70 and Ma-HSP70 are induced for 7h. at 25℃ and 30℃, respectively. In this study, two higher purified fusion protein of Ma-HSC70 and Ma-HSP70 have been obtained by prokaryotic expression of two HSP70s of Wuchang bream, thus laying foundation for further research on molecular structure and function of two proteins and associated antibodies. |
| Key words: Wuchang bream Megalobrama amblycephala, Heat shock protein 70, Prokaryotic expression, Fusion protein, Purification, Identification |
