含绿色荧光蛋白基因重组质粒的构建及其在血管平滑肌细胞中的表达

Construction of recombinant plasmid expressing green fluorescent protein and its expression in vascular smooth muscle cells

目的构建与增强型绿色荧光蛋白(GFP)融合的重组真核表达载体,并转染大鼠血管平滑肌细胞(A7r5),观察其在A7r5中的表达。方法采用Trizol法快速提取A7r5的总RNA,经RT-PCR获得线粒体融合蛋白2(mfn2)的cDNA,扩增、纯化、回收mfn2基因片段。用HindⅢ和BamHⅠ双酶切扩增的大鼠mfn2基因片段和质粒pEGFP-N1,然后将这2种酶切产物按常规方法连接、转化大肠杆菌DH5α。将提取的重组质粒进行酶切鉴定和测序。测序正确的重组质粒用脂质体法转染A7r5,24 h后观察GFP表达情况,并用RT-PCR、Western blot方法检测mfn2的表达。结果扩增出了1条约2.2 kb的片段,酶切结果显示重组质粒被切成4.7kb大小的pEGFP-N1载体和2.2kb大小的目的片段。经测序目的片段的序列与GeneBank中大鼠mfn2基因的开放阅读编码框序列完全一致。细胞转染48h后GFP表达量最多,RT-PCR、Western blot方法检测到mfn2在A7r5中高表达。结论成功构建了含GFP基因重组真核表达载体,并且可以在A7r5中高表达。

【Objective】 To construct an enhanced green fluorescent protein（GFP）labeled eukaryotic expression plasmid with mfn2 and to detect its expression in rat vascular smooth muscle cells（A7r5）.【Methods】 The RNA was extracted from A7r5 with efficient Trizol reagent.The mfn2 cDNA was obtained from it by reverse transcription polymerase chain reaction（RT-PCR） and was amplified,purified and retrieved.To digest pEGFP-N1 plasmid and mfn2 gene with dual-endonuclease,we retrieved a big fragment from pEGFP-N1 and a 2.2 kb fragment from mfn2 gene.Then the correctness of the pEGFP-mfn2 was evaluated by using restriction enzyme and sequencing analysis.The plasmid was transfected into the A7r5 with lipofectin,the transient expression of GFP was observed under fluorescence microscope after 24 hours and detected by RT-PCR,Western blot.【Results】 Correct construction of pEGFP-mfn2 was identified by methods of restriction enzyme analysis and nucleotide sequence determination.A total of about 70% transfected cells emitted out green fluorescence protein under fluorescent microscope after 48 h of transfection.The mfn2 gene expressed by the transfected cells were testified by RT-PCR,Western blot.【Conclusions】 The recombinant eukaryotic expression vectors have been constructed successfully and effectively expressed in A7r5.