含脂氧素A_4受体同源基因真核表达载体的构建与转染系膜细胞

Construction of Eukaryotic Expression Vector Containing Lipoxin A_4 Receptor Homologue Gene and Its Transfection into Mesangial Cells

目的:构建小鼠脂氧素A4受体同源基因(LRHG)的真核表达载体,克隆后转染小鼠肾小球系膜细胞,观察LRHG编码的脂氧素A4受体样蛋白(LRLP)在系膜细胞中的表达。方法:应用PCR方法,以小鼠睾丸cDNA为模板,扩增出LRHG基因片段,插入质粒pGEMT中,转化入大肠杆菌JM109。扩增阳性重组质粒,经酶切后纯化并测序鉴定目的片段。构建含6×组氨酸(His)基因的真核表达载体pcDNA3.1/LRHGhis,转化入大肠杆菌JM109扩增,酶切后再测序鉴定目的片段,抽提质粒后转染系膜细胞,进行Westernblot鉴定LRLP的表达。结果:测序鉴定表明,克隆的LRHG序列与GenBank中LRHG原序列100%符合,应用抗6×His抗体进行Westernblot鉴定系膜细胞中有LRLP的表达。结论:成功地构建了真核表达载体pcDNA3.1/LRHGhis,并转染了肾小球系膜细胞,获得了稳定的表达。

Objective: To construct and clone the eukaryotic expression vector of lipoxin A_4receptor homologue (LRHG), and to study the transfection and expression of the lipoxin A_4receptor-like protein (LRLP) in glomerular mesangial cells. Methods: The LRHG was amplified by polymerase chain reaction (PCR) from cDNA of mouse testis as template. The LRHG was inserted to the plasmid pGEM-T and then transformed into E.coli JM109. Positive clones were amplified, digested by restriction endonucleases, screened and identified by sequencing. The eukaryotic expression vector pcDNA3.1/LRHG containing 6×histidine gene was constructed, transformed into E.coli JM109, amplified and digested by restriction endonucleases. The sequence of inserted fragment in pcDNA3.1/LRHG was then analyzed. The transfection of the plasmid into mesangial cells was performed and identified by Western blot. Results: Sequence analysis showed that the gene homology between cloned LRHG and reported LRHG by GenBank was 100%. The expression of LRLP in mesangial cells was demonstrated by Western blot using the antibodies against 6×histidine. Conclusion: The eukaryotic expression vector pcDNA3.1/LRHG has been constructed successfully. The transfection of the plasmid into mesangial cells was performed successfully.