乳腺珠蛋白基因的克隆及重组蛋白的表达和纯化  

Clone of human mammaglobin gene as well as expression and purification of recombinant protein

作　　者：吴国球[1] 张臣[1] 孙宏伟[1] 赵成桂[1] 芦慧霞[1] 

机构地区：[1]东南大学附属中大医院临床检验中心,江苏南京210009

出　　处：《现代医学》2006年第2期71-75,共5页Modern Medical Journal

摘　　要：目的克隆人乳腺珠蛋白(hM aM)基因,获得高纯度hM aM重组蛋白。方法从人乳腺癌组织提取总RNA,逆转录合成cDNA,设计特异引物,用PCR方法扩增获得目的片段,利用T/A克隆将PCR产物插入pMD-18T载体。利用BamH I+XhoI双酶切方法将目的基因导入表达载体,阳性质粒转化BL21(DE3)感受态细胞,通过IPTG诱导获得重组蛋白,并用H is-SelectTM和Sephadex-G100进行纯化。结果用RT-PCR方法获得279 bp的片段,克隆至T载体后经DNA测序,结果与预期序列一致。表达质粒转化大肠杆菌后经诱导获得27 kD的目的蛋白,与预期分子质量一致。表达产物最终纯化为一条带。结论成功克隆hM aM基因,并获得高纯度重组蛋白,为hM aM的深入研究打下基础。Objective To clone human mammaglobin （hMaM） gene and obtain recombinant protein of hMaM with high purity. Methods Total RNA was extracted from breast cancer tissue and was reverse-transcripted into cDNA. The specific primers were designed for PCR to obtain the target gene fragment, and the fragment was inserted into pMD-18T vector by T/A match. The interested gene was connected with expressive vector by the method of double enzymes cut with BamHI ＋ XhoI. The positive plasmid was transformed into E. coli BL21 （DE3） competent cells. The recombinant protein was obtained by being induced with IPTG and purified by His-Select^TM and Sephadex-G100 chromatographies. Results The positive band of 279 bp showed up by RT-PCR and cloned to T-vector. Sequence result was in accordance with expected sequence. A 27 kD protein was obtained after expressive vector was transformed to E. coli BL21 （DE3）. Finally, the protein was purified to a single band and its molecular weight was expected. Conclusions A hMaM gene was successfully cloned, and recombinant protein with high purity was obtained. This study will be profound for the further research of human mammaglobin.

关 键 词：乳腺珠蛋白 乳腺癌 克隆 表达 

分 类 号：Q78[生物学—分子生物学] R737.9[医药卫生—肿瘤]