摘要
本试验旨在构建带有增强型绿色荧光蛋白报告基因EGFP及目的基因泛素化调节因子2(Smurf2)的真核表达载体pEGFP-N3-Smurf2,并转染小鼠肾小管上皮细胞检测其表达情况。用RT-PCR法扩增小鼠肾小管上皮细胞Smurf2的CDS区,构建克隆载体pEASY-Zero-Smurf2和真核表达载体pEGFP-N3-Smurf2,酶切并测序鉴定。运用脂质体转染法将pEGFP-N3-Smurf2真核表达载体转染小鼠肾小管上皮细胞,实时荧光定量PCR、免疫印迹检测Smurf2的表达。PCR结果显示扩增出Smurf2基因片段大小约为2247bp;重组质粒pEGFP-N3-Smurf2的酶切鉴定及测序结果均表明Smurf2基因成功克隆到真核表达载体中;脂质体转染后检测结果显示,转染pEGFP-N3-Smurf2表达载体的肾小管上皮细胞中Smurf2基因mR-NA表达量升高且EGFP-Smurf2重组蛋白有表达。本试验成功构建了含有增强型绿色荧光蛋白标记的Smurf2真核表达质粒,转染后能明显提高细胞内Smurf2的表达,为研究Smurf2基因的功能奠定了基础。
The assay was aimed to construct the eukaryotic expression vector pEGFP-N3-Smurf2 carrying Smad ubiquitina- tion regulatory factor 2 （Smurf2） and detect its expression after transfecting to renal tubular epithelial ceils （RTECs）. The CDS of Srnurf2 gene was amplified and cloned from total RNA of RTECs by RT-PCR. Both prokaryotie expression vector pEASY- Zero-Smurf2 and eukaryotic expression vector pEGFP-N3-Smurf2 were constructed and then detected by double digestion and sequencing, pEGFP-N3-Smurf2 was transfected into RTECs with LipofectamineTM 2000 and the expression of Smurf2 was de- tected by Real-time PCR and Western blotting. The amplified Srnurf2 gene was about 2247 bp in length. Restriction analysis and sequencing proved that Smurf2 was successfully inserted into recombinant plasmid pEGFP-N3. The mRNA and protein of Smurf2 were detected by Real-time PCR and Western blotting. Smurf2 expression vector using green fluorescent protein as a re- porter gene was constructed and transfected into RTECs successfully, which provided a powerful tool for the further study of function of Srnurf2 gene.
出处
《中国畜牧兽医》
CAS
北大核心
2013年第6期37-40,共4页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31172362
30972215)
北京市自然科学基金(5102014)
北京市人才强教深化计划(教学创新人才)
