人脂联素基因的克隆构建及其序列分析

Cloning and sequence analysis of human adiponectin gene

背景:脂联素已经成为缺血性疾病基因治疗的新靶点,而成功进行脂联素基因治疗的关键是脂联素基因的克隆。基因克隆主要有定向克隆法和T-A克隆法,定向克隆法操作较复杂;而T-A克隆法操作简便,成功率高。目的:应用T-A克隆法克隆人脂联素基因编码区,对其进行测序验证,并与GenBank比对。设计、时间及地点:基因水平的验证实验,于2006-06/2008-12在福建医科大学附属第一医院高血压研究所及福建中医学院中西医结合研究院完成。材料:脂肪组织取自福建医科大学第一附属医院外科患者术中切除的大网膜脂肪垫,液氮中冻存。Trizol为Invitrogen公司产品;M-MLV,Ge lExtract Kit为PROMEGA公司产品;Taq酶为TIANGEN公司产品;限制性内切酶BamHⅠ,SalⅠ,pMD18-T载体为TAKARA公司产品。方法:从人大网膜脂肪组织提取总RNA,经反转录-聚合酶链反应扩增出人脂联素编码区基因,再将人脂联素基因编码区克隆入载体pMD18-T中,通过限制性内切酶酶切鉴定后,对其进行测序验证,并与GenBank比对。主要观察指标:人脂联素克隆质粒酶切鉴定结果,人脂联素基因克隆序列与GenBank比对结果。结果:扩增得到人脂联素基因编码区,获得人脂联素的正向和反向克隆,与GenBank中人脂联素基因编码区序列比对均为完全一致。结论:成功地克隆出人脂联素基因编码区。

BACKGROUND： Adiponectin （apM1） has been a new target for gene therapy of ischemic diseases; apM1 gene cloning is the key of successful apM1 gene therapy. There are two main gene cloning methods： directional cloning and T-A cloning method. Directional cloning method is complicated, while the T-A cloning method is relatively simpler, with higher successful rate. OBJECTIVE： T-A cloning of apM1 gene coding region was applied to verify the sequence in comparison with GenBank. DESIGN, TIME AND SETTING： The gene verification experiment was performed at the laboratories of Fujian Hypertension Research Institute which is in The First Affiliated Hospital of Fujian Medical University and Academy of Integrative Medicine which is in Fujian University of Traditional Chinese Medicine, from June 2006 to December 2008. MATERIALS： Adipose tissue sample of omental fat pad was obtained from a surgical patient in the First Affiliated Hospital of Fujian Medical University. Trizol produced by Invitrogen company; M^-MLV, Gel Extract Kit produced by PROMEGA company; Taq enzyme produced by TIANGEN company; Restriction Endonucleases BamH I and Sal I, pMD18-T Vector produced by TAKARAcompany were used in this study. METHODS： Total mRNA was extracted from human greater ornentum adipose tissue. The coding region of human apM1 （hapM1） gene was amplified by RT-PCR. The coding region of hapM1 gene was cloned into pMD18-T vector .The recombinant plasmid was identified with restriction enzyme digestion analysis and nucleotide sequencing. MAIN OUTCOME MEASURES： The electrophoresis verification of the recombinant plasmids coding target gene using double enzyme digestion, comparison of the similarity between the sequences of the plasmids and hapM1 in GenBank. RESULTS： Sense and antisense coding region of hapM1 gene were cloned. The sequencing results showed that the sequences of the cloned DNA were completely identical to that of hapM1 in GenBank. CONCLUSION： The coding region of hapM1 gene was successfully cloned.