DPP-rhBMP-4m融合表达载体的构建和应用  

Construction and application of DPP-rhBMP-4m fusion protein expression vector

作　　者：王丽[1] 陈苏民[1] 陈南春[1] 张晓楠[1] 赵玮钦[1] 黄勇[1] 王涛[1] 关路媛[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033

出　　处：《第四军医大学学报》2007年第4期317-320,共4页Journal of the Fourth Military Medical University

基　　金：全军医药卫生科研基金(06MA230)

摘　　要：目的:构建容易去除标签蛋白的大肠杆菌融合蛋白表达载体.方法:PCR方法获得带有Asp-Pro-Pro前缀的重组人骨形态发生蛋白-4成熟肽(recombination human bone morphogenetic protein 4 mature peptide,rhBMP-4m)编码序列并克隆入pRSET载体,转化大肠杆菌后诱导表达;经免疫印迹法鉴定融合蛋白;甲酸裂解去除6His标签后细胞学实验检测其活性.结果:构建的融合表达载体序列测定结果与预期结果一致;诱导表达的DPP-rhBMP-4m融合蛋白以包涵体形式存在,占菌体总蛋白量的37%,亲和层析后目的蛋白纯度为97%;免疫印迹法鉴定显示,该融合蛋白可与相应抗血清产生特异性反应;对C2C12细胞具有良好的生物活性.结论:成功构建了利于目的蛋白纯化的pRSET-DPP-rhBMP-4m融合表达载体.AIM： To construct a E. coli fusion protein expression vector from which tag protein can be removed easily. METHODS： The codons of Asp-Pro-Pro were added to 5＇-teminal of rhBMP-4m （ recombination human bone morphogenetic protein 4 mature peptide） gene by PCR. The DPP- rhBMP-4m coding sequence was cloned into E. coli BL21 and induced to express with LPTG. The expressed fusion protein （DPP- rhBMP-4m） was analyzod by SDS-PAGE and Western Blot. After the 6His tag protein was removed by formic acid, the purified PP- rhBMP-4m was added to C2C12 cells to observe its biological activity. RESULTS： The DNA sequencing showed that the sequence of the fusion protein expression vector constructed in this study was exactly consistent with the one predicted. The fusion protein occupied 37% of the total bacteria protein, while the purity of the target protein（rhBMP-4m） reached 97% after affinity chromatography. And the protein could specifically react with antiserum and showed a good biological activity in C2C12 cells. CONCLUSION： The pRSET-DPP-rhBMP-4m vector is successfully constructed for the purification of interest protein.

关 键 词：重组人骨形态发生蛋白4 亲和层析 纯化 

分 类 号：R512.91[医药卫生—内科学]