摘要
以从枣树生长早期结果枝cDNA小规模测序获得的含2型金属硫蛋白基因的重组质粒pSPORT1-MT为模板,用PCR方法扩增出MT基因241bp特异片段,将其亚克隆入pBlue-scriptⅡSK-T载体,用限制性内切酶BamHⅠ和HindⅢ分别酶切重组pBluescriptⅡSK-T质粒和原核表达载体pET28a,然后将MT基因片段和表达载体pET28a通过T4DNA连接酶进行定向连接,用限制性内切酶BamHⅠ和HindⅢ酶切和PCR方法双重鉴定重组表达质粒。以pSPORT1-MT为模板扩增出大小为241 bp的片段,此片段与pBluescriptⅡSK-T载体连接后得到了亚克隆pBluescriptIⅡSK-MT载体,酶切回收了目的片段MT后,再次pET28a连接,得到了pET28a-MT原核表达载体。该重组表达载体经BamHⅠ和HindⅢ双酶切后,切出了235bp的目的片段,证明MT基因原核表达载体构建成功。
The DNA fragment of pSpoRT1-MT was amplified from the cDNA library of jujube by PCR, and subcloned into pBluescriptI Ⅱ SK-T vector. Then,the gene fragment of MT was obtained by the digestion of recombined pBluescript Ⅱ SK-T plasmid with endonuclease BamH Ⅰ and Hind Ⅲ. The prokaryotic expression vector pET28a was digested with the same endonucleases. The gene fragment of MT was orienedly linked into vector pET28a by T4 DNA ligase,and the recombined expression plasmid was identified by digestion analysis of endonuclease BamH Ⅰ and HindⅢand PCR gel electrophoresis analysis. The molecular size of PCR products was about 241 bp,as measured by gel electrophoresis analysis. Subcloned pBluescriptI Ⅱ SK-MT was obtained from pBluescript Ⅰ/SK-T and MT fragment. After destination fragment was restricted with endonuclease BamH I and HindⅢ and recovered, it was connected with pET28 by T4 DNA ligase to give pET28a-MT. The recombined expression plasmid pET28a-MT was digested into two fragments of about 235 bp and 5400 bp by endonuclease BamHⅠ and HindⅢ.
出处
《太原理工大学学报》
CAS
北大核心
2009年第4期388-390,共3页
Journal of Taiyuan University of Technology
基金
太原市科技项目明星专项(08121010)
山西省自然科学基金资助项目(20051081,20080011062-2)
作者简介
韩彩云（1980-），女，山西忻州人，硕士生，主要从事污水处理方面的研究，（Tel）13934580132
通讯联系人：曹秋芬，研究员，（Tel）0351-7127503
