中国组织工程研究Issue(2)：315-319,5.DOI:10.3969/j.issn.2095-4344.2013.02.023
突变型Cdh1基因真核表达质粒的构建及鉴定*☆
Construction and identification of a mutated-Cdh1 eukaryotic expressing vector
摘要
Abstract
BACKGROUND:The activity of anaphase promoting complex-Cdh1 is regulated by phosphorylation. Phosphorylated Cdh1 cannot be combined with anaphase promoting complex, thereby inhibiting the activity of the anaphase promoting complex. OBJECTIVE:To construct and identify a mutated-Cdh1 eukaryotic expressing vector. METHODS:The entire coding sequence of the Cdh1 gene was amplified from rat hippocampal mRNA by reverse transcription-PCR. Then the PCR product of Cdh1 was cloned into pBluescript plasmid by double digestion with restriction endonucleases EcoR1 and Xba1, and ligation. Based on site-directed mutagenesis, the pBluescript-Cdh1 plasmid containing Cdh1 coding sequence was used as a template. The 40, 151, 163 serine (S) and 121 threonine (T) in Cdh1 gene was mutated to alanine (A) by multiple PCR with four pairs of mutated primers. The mutated Cdh1 vector was identified by DNA sequencing. RESULTS AND CONCLUSION:The PCR product was about 1 500 bp by electrophoresis, including the entire coding sequence of Cdh1, restriction sites at both ends of Cdh1 and KOZAK sequence. The recombinant pBluescript-Cdh1 plasmid was identified by digestion with restriction endonuclease EcoR1 and Xba1, which was consistent with the expected results. DNA sequencing showed that A at the 930th base of Cdh1 (BC162059.1) coding sequence was mutated to G in the recombinant plasmid of pBluescript-Cdh1. But the sequence of amino acids was not affected. The 40, 121, 151, 163 amino acids in pBluescript-Cdh1-4A40, 121, 151, 163 mutant plasmids were al mutated to alanine. The mutated Cdh1 gene expressing plasmid at phosphorylation site was successful y constructed, which provides a good foundation for further studies of Cdh1 gene function.关键词
组织构建/组织构建细胞学实验/细胞周期末期促进复合物/Cdh1/定点突变/聚合酶联反应/真核表达质粒/神经系统/发育/神经损伤修复/基因治疗/国家自然科学基金分类
医药卫生引用本文复制引用
李立,石小云,张登文,张传汉,姚文龙..突变型Cdh1基因真核表达质粒的构建及鉴定*☆[J].中国组织工程研究,2013,(2):315-319,5.基金项目
国家自然科学基金(81000476)。 （81000476）
