大鼠β细胞素基因的克隆及其在大肠杆菌中的表达 |
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引用本文: | 安家泽,宋振顺,窦科峰,李开宗,韩骅. 大鼠β细胞素基因的克隆及其在大肠杆菌中的表达[J]. 医学争鸣, 2004, 25(23): 2141-2143 |
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作者姓名: | 安家泽 宋振顺 窦科峰 李开宗 韩骅 |
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作者单位: | 第四军医大学西京医院肝胆外科,陕西,西安,710033;第四军医大学基础部医学遗传学与发育生物学教研室,陕西,西安,710033 |
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摘 要: | 目的: 获得大量重组大鼠β细胞素(BTC),为研究β细胞素的功能及制备其抗体奠定基础. 方法: 利用PCR方法从大鼠肾组织扩增出544 bp的β细胞素基因片段并按读框克隆到原核表达载体pET28a( )上,得到重组质粒pET28a-rBTC,转化大肠杆菌BL-21(DE3)菌株,IPTG诱导β细胞素蛋白表达,SDS-PAGE 和Western blot检测. 结果: 经IPTG诱导后,有明显的目的蛋白表达,分子量符合β细胞素;表达的蛋白主要以包涵体形式存在;表达量约占菌体蛋白总量的20%~30%;目的蛋白与抗His标签抗体具有良好的反应原性. 结论: 大鼠β细胞素基因的克隆与表达为进一步开展β细胞素蛋白功能和胰腺干细胞的研究奠定了基础.
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关 键 词: | β细胞素 基因克隆 基因表达 pET载体 |
文章编号: | 1000-2790(2004)23-2141-03 |
修稿时间: | 2004-06-16 |
Cloning of rat betacellulin gene and its expression in E.coli |
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Abstract: | AIM: To obtain large amount of rat betacellulin so as to explore the function of the protein and prepare the antibody against this protein. METHODS: A 544 bp of rat betacellulin gene fragment was amplified by PCR method from rat kidney and cloned into pET28a(+)vector, an E.coli expression vector, to construct a recombinant plasmid pET28a-rBTC. The plasmid was transformed into E.coli BL-21 (DE3) and induced to express betacellulin with IPTG. The expression of betacellulin was detected by SDS-PAGE electrophoresis and Western blot. RESULTS: A novel protein with expected molecular weight was expressed upon induction with IPTG. The expressed product showed good reactivity to anti-His tag antibody, and was most in inclusion body sections. CONCLUSION: Cloning and expression of betacellulin gene lay a basis for the further study on the function of this protein and pancreatic stem cell differentiation. |
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Keywords: | Betacellulin gene clone gene expression pET vector |
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