杜氏盐藻核基质附着区结合蛋白基因的克隆及原核表达  

Cloning and prokaryotic expression of matrix attachment region binding protein gene of Dunaliella salina

作　　者：冯英才[1] 王天云[2] 王鹏举[1] 刘红涛[1] 刘兰琦[1] 薛乐勋[1] 

机构地区：[1]郑州大学生物工程系细胞生物学研究室,郑州450052 [2]新乡医学院生物化学与分子生物学教研室,新乡453003

出　　处：《郑州大学学报（医学版）》2008年第6期1120-1123,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金资助项目30470030

摘　　要：目的:构建杜氏盐藻核基质附着区(MAR)结合蛋白的原核表达载体,转化大肠杆菌BL21(DE3),表达并纯化融合蛋白。方法:根据GenBank上杜氏盐藻MAR结合蛋白的cDNA全长序列设计出含有特定酶切位点的引物,PCR法获得MAR结合蛋白基因序列全长,将其克隆至载体pMD18-T中。酶切连接到pET-28a(+)上构建原核表达载体pET-MBP,转化感受态大肠杆菌BL21(DE3),异丙基硫代-β-半乳糖苷(IPTG)诱导其表达,Western blotting检验其表达结果。结果:重组质粒经酶切、PCR、测序鉴定,成功构建了原核表达载体pET-MBP,对表达的融合蛋白的Western blotting分析结果表明表达的蛋白质为目的蛋白。Ni2+亲和层析法纯化蛋白质,纯化后目的蛋白在总蛋白中的含量约为70%。结论:成功地克隆杜氏盐藻MAR结合蛋白基因的全长并在大肠杆菌BL21(DE3)中表达,且纯化了MAR结合蛋白的融合蛋白。Aim： To express matrix attachment region（MAR） binding protein（MBP） of Dunaliella salina in the prokaryotic expression system.Methods：The MBP gene was amplified by RT-PCR using a pair of primers designed according to the cDNA sequence of MBP gene with specific restriction enzyme sites,followed ligated into the vector pET28（＋） to construct a recombinant vector pET-MBP,then transformed into E.coli BL21（DE3） to express the fusion protein with the presence of IPTG.Western blotting was performed to determine the expression of the fusion protein.The protein was purified by Ni^2＋ affinity chromatography.Results： The results showed that RT-PCR products were about 1 600 bp long,the fusion protein was about 75 000 measured by SDS-PAGE.The fusion proteins accounted for more than 70% of the total protein.Conclusion：The prokaryotic expression vector has been constructed successfully.

关 键 词：杜氏盐藻 核基质附着区 核基质附着区结合蛋白 原核表达 

分 类 号：Q781[生物学—分子生物学]