mcpr1基因原核表达载体的构建及其在大肠杆菌中的表达 |
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引用本文: | 轩东英,金岩,金明,轩昆,张蓉.mcpr1基因原核表达载体的构建及其在大肠杆菌中的表达[J].实用口腔医学杂志,2004,20(5):554-557. |
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作者姓名: | 轩东英 金岩 金明 轩昆 张蓉 |
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作者单位: | 1. 第四军医大学口腔医学院组织工程实验中心,710032 2. 第四军医大学口腔医学院生物化学教研室,710032 3. 第四军医大学口腔医学院药理教研室,710032 |
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基金项目: | 西安第四军医大学创新基金重点课题资助CXO2F0 0 2 |
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摘 要: | 目的 :构建mcpr1基因原核表达载体 ,表达MCPR1蛋白。 方法 :采用多聚酶链式反应 (PCR)扩增出mcpr1基因的蛋白编码序列 ,克隆到中间载体中 ,再经双酶切 ,亚克隆到表达载体中 ,构建该基因的融合表达载体pGEX 4T mcpr1,在大肠杆菌中用IPTG诱导表达 ,SDS PAGE电泳。 结果 :酶切和测序鉴定 ,表明融合表达载体内插入片段正确 ,在大肠杆菌中诱导后 ,出现一条 3 60 0 0的新蛋白带 ,主要存在于细菌沉淀中 ,占总蛋白的3 9 %。结论 :研究表明mcpr1基因编码的蛋白质能够在原核细胞中表达 ,为进一步深入研究该蛋白质的结构与功能打下基础
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关 键 词: | mcpr1基因 基因表达 融合蛋白 蛋白纯化 |
文章编号: | 1001-3733(2004)-05-0554-04 |
修稿时间: | 2003年12月23 |
Construction of mcpr1 gene vector and expression of mcpr1 in escherichia coli |
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Abstract: | Objective: To construct mcpr1prokaryoti c expression vector and to express MCPR1 protein.Methods:PCR was used to obtain coding region of mcpr1. Construction of a high-level fusion protein expression vector pGEX-4T-mcpr1 was conducted by inserting the fra gment of coding region of mcpr1into a fusion protein expression vector pGEX -4T-1. Then the recombinant plasmid was transferred into E. colito prepar e the MCPR1/GST fusion protein. DNA sequencing and endonucleases digesting were used to check the coding region. Results:pGEX-4T- mcpr1 wa s constructed successfully and the coding region was inserted into the vector co rrectly. A new protein band of 36 000 was observed by SDS-PAGE analysis after i nduction by IPTG. The 36 000 protein amounted to 39 percent of the total prote in and existed mostly in precipitation of broken bacteria. Conclusion: MCPR1 protein can be expressed in E. coliexpression system and purif ied initially. |
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Keywords: | mcpr1 gene Fusion protein Gene expression Purifi cation |
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