摘要
背景:新近的一些研究认为,采用体外重组瘦素替代疗法对遗传性肥胖、难愈性创面和内源性瘦素缺乏应是行之有效的方法。目的:取人脂肪组织获取瘦素基因,构建pET28a-leptin重组子。设计、时间及地点:单一样本观察,实验于2004-06/2007-12在江西省医学科学研究所抗肿瘤室完成。材料:取自22岁汉族女性行重睑手术时皮下脂肪组织,冻存于液氮中备用,供者知情同意。方法:取人脂肪组织,反转录-聚合酶链反应得到瘦素基因后,通过DNA重组技术克隆至pMD18T载体与pET28a原核表达载体,EcoRⅠ+XhoⅠ双酶切及测序鉴定。主要观察指标:①cDNA扩增结果。②重组质粒鉴定。③DNA测序结果。结果:扩增得到520bp目的基因;所构建重组瘦素原核表达载体pET28a-leptin经双酶切电泳见520bp目的条带,DNA测序与相应基因序列同源性达100%。结论:通过DNA重组至pET28a原核表达载体,经EcoRⅠ+XhoⅠ双酶切及测序鉴定成功构建pET28a-leptin重组子。
BACKGROUND： Previous studies consider that recombination leptin in vitro is effective method in treating with genetic obesity refractory wound, or endogenous leptin deficiency. OBJECTIVE： To construct pET28a-leptin recon by leptin gene from human adipose tissue. DESIGN, TIME AND SETTING： A single sample observation was conducted at Department of Oncology, Jiangxi Institute of Medical Sciences from May 2004 to December 2007. MATERIALS： The subcutaneous adipose tissue was obtained from a female aged 22-year-old, who underwent double eyelid operation. The informed consent was obtained from the donor. METHODS： Human adipose tissue was used for amplifying leptin gene by RT-PCR. The gene was cloned into pMD18T and pET28a to construct recombinant, which was identified by double enzyme digest and DNA sequencing. MAIN OUTCOME MEASRES： The cDNA amplified, recombinant plasmid identified and DNA sequence were observed. RESULTS： The fragment of 520 bp was obtained. Target band with 500 bp could be found in the prokaryotic expression vector pET28a-leptin after double enzyme digest, which 100% identified with that reported in Genbank. CONCLUSION： The recombinant pET28a-leptin can be constructed by recombinant DNA to pET28a prokaryotic expression vector, followed by EcoRⅠ[ ＋Xho ] double enzyme digestion.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第7期1317-1319,共3页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
江西省卫生厅重大招标计划(200307)资助~~
