人regucalcin和红色荧光蛋白融合基因的构建及表达

Construction and expression of human regucalcin fused with red fluorescent protein gene

目的:构建人regucalcin(RGN)全长cDNA的真核表达载体pcDNA3.1-RGN-DsRed-zeo,观察RGN及红色荧光蛋白(DsRed)在人肾皮质近曲小管上皮细胞(HK-2细胞)中的表达。方法:RT-PCR技术扩增获得人HK-2细胞RGN全长cNDA,将扩增的产物进行T-A克隆,经酶切、DNA测序鉴定正确,将RGN基因酶切后构成pDsRed-RGN,再将RGN基因与DsRed一起酶切构建成pcDNA3.1-RGN-DsRed-zeo的真核表达重组质粒。用脂质体将重组质粒转染入HK-2细胞,激光共聚焦显微镜观察重组质粒的表达情况。结果:RT-PCR扩增获得人HK-2细胞,其RGNcDNA全长897bp,构建完成真核表达重组质粒pcDNA3.1-RGN-DsRed-zeo,经DNA测序与GenBank中的人RGNcDNA序列一致。经zeocin(zeo)筛选获得稳定表达重组质粒的单克隆细胞株。激光共聚焦显微镜观察到转染重组质粒的细胞中有红色荧光蛋白的表达。结论:本实验成功构建了pcDNA3.1-RGN-DsRed-zeo真核表达质粒,并发现其在HK-2细胞中表达。

Objective To construct a fused gene of human regucalcin（RGN） and red fluorescent protein （pcD- NA3.1-RGN-DsRed-zeo）, and to detect its expression in human kidney 2（HK-2） cell. Methods The full-length cDNA encoding RGN gene was amplified by reverse transcription polymerase chain reaction （RT-PCR） and subcloned to eukaryotic expression vector pDsRed-N1. The recombinant pDsRed-RGN was transformed into E.coli.DH5α, which was identified by double digestion with restriction endonucleases and sequencing, and then RGN-DsRed were retransformed into pcDNA 3.1 （＋）-zeo by restriction endonucleases. The recombinant pcDNA3.1-RGN-DsRed-zeo was transformed into E.coli.DH5α, which was identified by double digestion with restriction endonucleases and sequencing. The sequence of RGN full-length cDNA was confirmed by Genbank. The recombinant plasmids were transfected into HK-2 cells by lipofectamine method. The RGN mRNA expression in HK-2 cells was assayed by laser scanning confocal microscopy （LSCM）. Results The sequence of RGN cDNA was identical with that in Genbank. The RGN fusion proteins were expressed in the cells transfected with pcDNA3.1-RGN-DsRed-zeo plasmid. Red fluorescence was observed by LSCM on the cells transfected with pcDNA3.1-RGN-DsRed-zeo plasmid. Conclusions The recombinant eukaryotic expression vector pcDNA3.1-RGN-DsRed-zeo is successfully constructed and has expression in the HK-2 ceils.