学术
摘 要:
目的构建大鼠肾上腺髓质素（ADM）真核表达质粒,观察其在大鼠近端肾小管上皮细胞（NRK-52E）中的表达。方法提取大鼠肾上腺组织总RNA,RT-PCR扩增ADM全长cDNA;将扩增产物连接于pMT-18T载体,双酶切及测序鉴定重组质粒。用限制性内切酶BamHⅠ/EcoRⅠ分别对pMT-18T-ADM和真核表达质粒pcDNA3.1双酶切;将目的基因定向克隆到pcDNA3.1载体上;对重组质粒进行双酶切鉴定。将重组质粒通过脂质体介导转染NRK-52E,RT-PCR检测ADM表达。结果特异扩增出ADM片段,大小为585 bp;片段成功插入pcDNA3.1载体中。RT-PCR方法证明NRK-52E中存在ADM高表达。结论成功构建大鼠ADM真核表达质粒,并将其成功转染NRK-52E。[著者文摘]
文章出处:
《山西医科大学学报》-2007年38卷10期 -899-902页
栏目信息:
分 类 号:
文献标识码:
A
文章编号:
1007-6611（2007）10-0899-04
Construction of recombinant plasmid pcDNA3.1-adrenomedullin and its expression in rat renal epithelial cells
QIAO Xi, WANG Li-hua, LI Rong-shan, et al （ Dept of Nephrology, Second Clinical Medical College, Shanxi Medical University, Taiyuan 030001, China ）
Abstract:
Objective To construct a eukaryotic expression plasmid pcDNA3.1-adrenomedullin（pcDNA3.1-ADM）,and to explore its expression in rat renal epithelial cells（NRK-52E）.Methods The total RNA was extracted from the adrenal glands in Wistar rats.The full-length cDNA of rat ADM was amplified by RT-PCR and cloned into pMT-18T vector using TA cloning,and then identified with double digestion and sequencing.The pMT-18TADM and plasmid pcDNA3.1 were double digested with BamHⅠ and EcoRⅠ respectively,and the target gene was cloned into pcDNA3.1 directionally.The recombinant plasmid pcDNA3.1-ADM was identified by double digestion,and then transected into NRK-52E by liposome.Expression of ADM in pcDNA3.1-ADM transferred cells were detected by RT-PCR.Results The destination gene,585 bp,was distinctively amplified by PCR,and inserted successfully into the plasmid pcDNA3.1.Expression of ADM in cells transfected with pcDNA3.1-ADM was much higher than in controls.Conclusion The eukaryotic expression plasmid pcDNA3.1-ADM is successfully constructed and expressed in NRK-52E.[著者文摘]
Key words:
adrenomedullin; gene clone; eukaryotic expression; rats
基金资助:
山西医科大学博士启动基金资助项目（200625）;山西医科大学第二临床医学院博士启动基金资助项目（2006-08）
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