| 摘要: |
| 【目的】对康氏木霉HEX1蛋白进行原核表达和纯化，为进行抗体制备及深入研究HEX1功能奠定基础。【方法】采用RT-PCR方法扩增康氏木霉hex1基因，构建pMD19-T-hex1重组克隆质粒，在此基础上构建pET 28a-hex1表达载体，转化E.coli BL21（DE3）细胞。通过IPTG诱导表达，对重组HEX1蛋白进行可溶性分析、纯化及鉴定。【结果】成功克隆了长660 bp的hex1基因并构建了融合表达载体，HEX1蛋白在IPTG 诱导下得到表达。融合蛋白主要以包涵体形式存在，能与抗His标签的兔多克隆抗体发生特异性反应，并纯化出了融合表达蛋白。【结论】康氏木霉HEX1蛋白在原核细胞中得到成功表达及纯化，获得了分子质量约为25 ku的重组蛋白。 |
| 关键词: 康氏木霉 HEX1 原核表达 蛋白纯化 |
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| 基金项目:国家自然科学基金项目（41001189）；安徽省高校自然科学研究重点项目（KJ2013A203）；阜阳师范学院科技成果孵化基金项目（2013KJFH04）；阜阳师范学院教研项目（2013JYXM30） |
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| Prokaryotic expression and purification of HEX1 of Trichoderma knoningii |
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TANG Jun,LU Juan,XU Hui-fen,et al
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| Abstract: |
| 【Objective】The paper aimed to express and purify HEX1 in Escherichia coli,which would lay basis for producing antibody and exploring the function of HEX1.【Method】The hex1 gene was amplified by RT-PCR and recombinant plasmid pMD19-T-hex1 was constructed.Then the recombinant expression plasmid pET28a-hex1 was constructed and transformed into E.coli BL21 (DE3) for expression of HEX1 induced by IPTG.The induced fusion protein was purified and verified by SDS-PAGE and Western blot.【Result】The hex1 gene (660 bp) was cloned and the recombinant vector was constructed.The expressed fusion proteins induced by IPTG were inclusion bodies.The fusion proteins with His tag were also purified.【Conclusion】HEX1 was successfully expressed and purified in prokaryotic cells,and the molecular weight of the recombinant protein was about 25 ku. |
| Key words: Trichoderma knoningii HEX1 prokaryotic expression protein purification |
