乳腺珠蛋白_____]乳腺珠蛋白；乳腺癌；克隆；表达

乳腺珠蛋白基因的克隆及重组蛋白的表达和纯化

http://www.100md.com 《现代医学》 2006年第2期

乳腺珠蛋白,,]乳腺珠蛋白；乳腺癌；克隆；表达,1材料和方法,2结果,3讨论,[参考文献]

     [摘要]目的克隆人乳腺珠蛋白(hMaM)基因，获得高纯度hMaM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BamHI+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HisSelectTM和SephadexG100进行纯化。结果用RTPCR方法获得279bp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27kD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hMaM基因，并获得高纯度重组蛋白，为hMaM的深入研究打下基础。

    [关键词]乳腺珠蛋白；乳腺癌；克隆；表达

    Clone of human mammaglobin gene as well as expression and

    purification of recombinant proteinWU Guoqiu,ZHANG Chen,SUN Hongwei,ZHAO Chenggui,LU Huixia

    (Center of Clinical Laboratory Medicine,Zhongda Hospital,Southeast University,Nanjing 210009,China)

    Abstract：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SelectTM 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. ......

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