中文摘要：

背景：实验表明，通过激活单磷酸腺苷激活的蛋白激酶（AMP—ActivatedProteinKinase，AMPK）α2可以增加胰岛素的敏感性和骨骼肌葡萄糖的摄取，其有望成为预防和治疗2型糖尿病的新的生理和药理作用靶点。目的：克隆人的AMPKa2基因，并构建其野生型和突变型真核表达载体。设计：单一样本观察。时间及地点：实验于2007—04／2008—01在中山大学附属第二医院I临床分子生物实验室完成。材料：QuikChangeIISite—DirectedMutagenesisKit为Stratagene公司产品。真核表达载体pcDNA3．1（＋），大肠杆菌DH5a为实验室保存。人骨胳肌组织来源于中山大学附属第二医院手术截肢患者，获患者知情同意，新鲜取材后液氮冷冻。方法：采用反转录-聚合酶链反应技术从人骨骼肌扩增AMPKα2基因，并将其克隆到T载体，通过测序对其进行鉴定。采用Quickchange定点诱变试剂盒对AMPKa2基因进行体外定点诱变，并将其野生和突变的编码基因亚克降到真核表达载体pcDNA3．1中，通过酶切和测序进行鉴定。主要观察指标：①目的基因的克隆。②定点诱变。③真核表达质粒的构建。结果：成功克隆了AMPKα2基因，大小约1700bp，与已发表的AMPKα2同源性为99％，GenBank录入号EF056019。成功将AMPKα2第45位Lysine（AAA）突变为Arginine（AGA），成功构建了野生型和突变型pcDNA—AMPKα2重组质粒。结论：实验成功克隆了AMPKα2基因，构建了其野生型和突变型真核表达载体。

英文摘要：

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 AMPKα2 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 Yat-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. coil DH5α 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 AMPKa2 subunit gene 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 carried 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： （1） The cloning of aim gene; （2） site-directed mutagenesis; （3） eukaryotic expression plasmid. RESULTS： The human AMPKα2 subunit gene （about 1 700 bp） was successfully cloned, with 99% homology to the reported AMPK α2 gene. A GenBank accession number was EF056019. The achieved mutation of the 45th Lysine （AAA） was found to Arginine（AGA）. The wild-type and mutant pcDNA- AMPKα2 recombinant plasmids were constructed su