摘要
目的克隆人p27kip1基因并构建其真核表达载体,为进一步研究其功能奠定基础。方法根据p27kip1已知序列设计引物,分别在上下游引入MluⅠ和SpeⅠ酶切位点,从人肾脏组织中提取总RNA,经逆转录PCR得到cDNA,亚克隆入携带GFP报告基因的pWPXL真核载体中,经筛选获得正确的克隆,并通过双酶切凝胶电泳及测序鉴定。将成功构建的质粒在脂质体介导下转染肾癌786-0细胞,并通过RT-PCR及Western-blot检测表达情况。结果克隆了人p27kip1基因,与GenBank中序列比对无误;真核表达载体经酶切鉴定表明为正确重组子,成功构建了真核表达载体。转染肾癌786-0细胞48h后,p27kip1蛋白出现高表达。结论成功克隆了p27kip1基因,构建了其真核表达载体并在肾癌细胞中成功表达,为p27kip1在肾肿瘤中的进一步研究提供了工作基础。
Objective To clone and construct eukaryotic expression vector carrying a human p27^kip1 gene. Method Total RNA was prepared from human renal tissue, p27^kip1 gene was amplified by reverse transcriptionpolymerase chain reaction （RT-PCR） with a pair of specific primers containing the Mlu I and Spe I restriction sites. PCR products were then subcloned into the eukaryotic expression vector pWPXL. Recombinant plasmids were identified by two restriction endonucleases analysis and sequencing. The 786-0 kidney tumour cell line was then transfected by the recombinant plasmid with lipofectamine. Expression of p27^kip1 in 786-0 cells was determined by RT- PCR and Western-blot. Result p27^kip1 gene was successfully cloned into the eukaryotic expression vector pWPXL and the sequence matched the sequence deposited in GenBank. Over-expression of p27^kip1 protein was observed in 786-0 cells at 48 hours after transfection with pWPXL-p27^kip1. Conclusion p27^kip1 gene was successfully cloned and expressed in 786-0 cells. The plasmid may be used for the study of renal carcinoma.
出处
《热带医学杂志》
CAS
2008年第4期285-287,305,共4页
Journal of Tropical Medicine
