中国组织工程研究与临床康复2009，Vol.13Issue(28)：5554-5557,4.DOI:10.3969/j.issn.1673-8225.2009.28.032
AMPKα2基因克隆及其野生型和突变型真核表达载体的构建
Cloning of activating adenosine monophosphate-activated protein kinase alpha 2 subunit gene and construction of its wild-type and mutant eukaryotic expression vectors
摘要
Abstract
BACKGROUND: The experimental results showed that insulin sensitivity and glucose uptake in skeletal muscle could be improved by activating adenosine monophosphate-activated protein kinase a2 (AMPKα2). AMPKa2 is expected to become a new physiological and pharmacological target for the prevention and treatment of type 2 diabetes mellitus. OBJECTIVE: To clone human AMPKa2 subunit gene and to construct its wild-type and mutant eukaryotic expression vectors. DESIGN: A single sample observation.TIME AND SETTING: The experiment was performed in the Clinical Molecular Biology Laboratory, the Second Affiliated Hospital of Sun Yet-sen University from April 2007 to January 2008.MATERIALS: QuikChange II Site-Directed Mutagenesis Kit was produced by Stratagene. Eukaryotic expression vector pcDNA3.1(+) and E. coll DH5a were provided by the laboratory. Human skeletal muscle tissue was from patients who received amputation surgery in the Second Affiliated Hospital of Sun Yat-sen University. Informed consent was obtained from the patients, and fresh samples were collected and frozen in liquid nitrogen.METHODS: The human AMPKo2 subunit gone was amplified from human skeletal muscle by RT-PCR, cloned into T vector, and the recombinant plasmid was confirmed by sequencing. In vitro site-directed mutagenesis was carded out with Quickchange site-directed mutagenesis kit. The wild-type and mutant coding genes were subcloned into eukaryotic expression vector pcDNA3.1, and the recombinant plasmids were validated by enzyme digestion and sequencing.MAIN OUTCOME MEASURES: ①The cloning of aim gone; ②site-directed mutagenesis; ③ eukaryotic expression plasmid. RESULTS: The human AMPKα2 subunit gene (about 1700 bp) was successfully cloned, with 99％ homology to the reported AMPK α2 gene. A GenBank accession number was EF056019, The achieved mutation of the 45<'th> Lysine (AAA) was found to Arginine(AGA). The wild-type and mutant pcDNA-AMPKα2 recombinant plasmids were constructed successfully. CONCLUSIONS: The human AMPKα2 subunit gene was cloned successfully from human skeletal muscle and its wild-type and mutant eukeryotic expression vectors were constructed successfully in the experiment.关键词
AMPKα2/克隆/突变/真核表达载体分类
医药卫生引用本文复制引用
罗招凡,李芳萍,丁鹤林,程桦..AMPKα2基因克隆及其野生型和突变型真核表达载体的构建[J].中国组织工程研究与临床康复,2009,13(28):5554-5557,4.基金项目
国家自然科学基金资助项目(30570886) （30570886）
a grant from the National Natural Science Foundation of China,No. 30570886 （）
