CAJ | 학술논문

背景:实验表明,通过激活单磷酸腺苷激活的蛋白激酶(AMP-Activated Protein Kinase, 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AMPKα2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临床分子生物实验室完成.材料:QuikChange Ⅱ Site-Directed Mutagenesis Kit为Stratagene公司产品.真核表达载体pcDNA3.1(+),大肠杆菌DH5α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:采用反转录一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AMPKα2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隆到真核表达载体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1 700 bp,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-AMPK α2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.
배경:실험표명,통과격활단린산선감격활적단백격매(AMP-Activated Protein Kinase, AMPK)α2가이증가이도소적민감성화골격기포도당적섭취,기유망성위예방화치료2형당뇨병적신적생리화약리작용파점.목적:극륭인적AMPKα2기인,병구건기야생형화돌변형진핵표체재체.설계:단일양본관찰.시간급지점:실험우2007-04/2008-01재중산대학부속제이의원림상분자생물실험실완성.재료:QuikChange Ⅱ Site-Directed Mutagenesis Kit위Stratagene공사산품.진핵표체재체pcDNA3.1(+),대장간균DH5α위실험실보존.인골각기조직래원우중산대학부속제이의원수술절지환자,획환자지정동의,신선취재후액담냉동.방법:채용반전록일취합매련반응기술종인골격기확증AMPKα2기인,병장기극륭도T재체,통과측서대기진행감정.채용Quickchange정점유변시제합대AMPKα2기인진행체외정점유변,병장기야생화돌변적편마기인아극륭도진핵표체재체pcDNA3.1중,통과매절화측서진행감정.주요관찰지표:①목적기인적극륭.②정점유변.③진핵표체질립적구건.결과:성공극륭료AMPKα2기인,대소약1 700 bp,여이발표적AMPKα2동원성위99%,GenBank록입호EF056019.성공장AMPKα2제45위Lysine(AAA)돌변위Arginine(AGA),성공구건료야생형화돌변형pcDNA-AMPK α2중조질립.결론:실험성공극륭료AMPKα2기인,구건료기야생형화돌변형진핵표체재체.
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.