摘要
目的:分离大鼠胰岛素基因增强结合蛋白1(Islet-1)基因,构建plEGFP-C1-Islet-1逆转录病毒表达载体。方法:利用RT-PCR技术钓取大鼠Islet-1基因,克隆到测序载体PGET-1TA中,测序后再亚克隆到逆转录病毒载体plEGFP-C1中,通过脂质体2000将其导入包装细胞PA317中,经G418筛选后,获得阳性克隆。结果:RT-PCR产物为1050bp条带,经测序鉴定与GenBank中Islet-1基因的序列相同;构建的plEGFP-C1-Islet-1载体经酶切鉴定,证实Islet-1基因片段正确插入逆转录表达载体中。用共聚焦激光扫描显微镜观察导入Islet-1基因的PA317细胞,可见细胞发出绿色荧光。结论:分离得到了大鼠Islet-1基因并成功构建了plEGFP-C1-Islet-1表达载体。
Objective To isolate the rat insulin gene enhancer binding protein 1 （Islet-1） gene and construct plEGFP-C1-Islet-1 recombinant retroviral expression vector. Methods The cDNA encoding the rat Islet-1 gene was isolated by RT-PCR method, the cDNA was first cloned into PGET-1 TA vector to facilitate the sequence and then subcloned into the retroviral vector plEGFP-C1, plEGFP-C1-Islet-1 was transfected into PA317 packaging cells with lipofectamine 2000. Transformants were selected in medium containing G418. Results A 1 050 bp DNA fragment was obtained by RT-PCR; plEGFP-C1-Islet-1 recombinant retroviral expression vector was identified by restrictive enzymes digestion, PA317 cells transfected with recombinant vector expressed enhancer green fluorescent protein （EGFP）. Conclusion The gene encoding the rat Islet-1 is obtained and plEGFP-C1-Islet-1 expression vector is constructed successfully.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2007年第5期831-833,共3页
Journal of Jilin University：Medicine Edition
基金
教育部高等学校博士学科点专项科研基金(20030183048)
吉林大学种子基金资助课题(2005)
