绿色荧光蛋白标记的人脂联素真核表达载体的构建

目的：构建绿色荧光蛋白（GFP）标记的人脂联素（Adiponectin）真核表达质粒。方法：用本实验室已构建好的人脂联素克隆载体pMD18—T／Adiponeetin为模板，结合已设计好的特异性引物。采用PCR方法克隆出Adiponectin目的片段，将该目的片段再连至真核表达载体pcDNA3．1／CT—GFP—TOPO上，转化入TOP10感受态细胞中，通过筛选得到融合有绿色荧光蛋白的重组质粒pcDNA3．1／CT—GFP—TOPO—Adiponectin。结果：PCR获得长度为736bp的目的片段，经pcDNA3．1／CT—GFP-TOPO真核表达载体连接、筛选及序列分析后，证实所插入的目的片段与genebank检索的人脂联素cDNA序列（Accession NM-004797）100％配。结论：绿色荧光蛋白标记的人脂联素真核表达载体构建成功。

Construction and Identification of Eukaryotic pcDNA3.1/CT- GFP-TOPO-Adiponectin Expression Vector

Objective: To construct the pcDNA3.1/CT -GFP-TOPO-Adiponectin expression vector. Methods: The encoding fragment of human Adiponectin gene was obtained from a recombinant plasmid pMD18-T/Adiponectin using PCR, then the gene was inserted into the vector pcDNA3.1/CT-GFP-TOPO after sequencing, which introduced into TOP10 competent cells in order to choose and sequence. Results: A 736 bp fragment was obtained by PCR clone after bloting and sequencing. The results were matching to the cDNA of human adiponectin, which accession of the gene bank was NM-004797. Conclusion: The eukaryotic pcDNA3.1/ CT-GFP-TOPO- Adipon ectin expression vector was constructed successfully.