人Regucalcin cDNA真核表达载体的构建及表达

Construction of human regucalcin eukaryotic expression vectors and expression

目的构建人regucalcin(RGN)全长cDNA的真核表达载体,观察其在人肾皮质近曲小管上皮细胞(HK-2细胞)中表达。方法用RT-PCR技术扩增获得人HK-2细胞RGN全长cNDA,将扩增的产物连接入pMD18-T克隆载体上,将重组质粒转化入大肠杆菌DH5α内并提取质粒,双酶切鉴定、DNA测序鉴定准确,再用双酶切方法将RGN基因定向连接到真核表达载体pEGFP-N1中,构成pEGFP-RGN,将pEGFP-RGN转化入大肠杆菌DH5α内并提取质粒,双酶切鉴定、DNA测序鉴定准确,再用双酶切方法将RGN基因与EGFP定向连接到真核表达载体pcDNA3.1(+)-zeocin中,构建真核表达pcDNA3.1-RGN-EGFP-zeo的重组体,将pcDNA3.1-RGN-EGFP-zeo转化入大肠杆菌DH5α内并提取质粒,双酶切鉴定、DNA测序鉴定准确,用脂质体转染入HK-2细胞,荧光显微镜、激光共聚焦显微镜观察pcDNA3.1-RGN-EGFP-zeo表达情况。结果RGN cDNA全长是897 bp,以此构建的pcDNA3.1-RGN-EGFP-zeo真核表达载体,经DNA测序与GenBank中的人RGN cDNA序列一致。荧光显微镜、激光共聚焦显微镜观察到pcDNA3.1-RGN-EGFP-zeo转染的细胞中有绿色荧光蛋白的表达。结论本实验成功构建了pcDNA3.1-RGN-EGFP-zeo真核表达质粒,并在HK-2细胞中表达。

Objective To construct the eukaryotie expression vector of human regucalcin（RGN）,and to detect their expressing in human HK-2 cell.Methods The full-length cDNA encoding RGN gone was amplified by reverse transcription polymerase chain reaction （RT-PCR） and subcloned to eukaryotic expression vector pEGFP-N1 ,the recombinant pEGFP-RGN was transformed into E. coli.DH5α,identified by double digestion with restriction endonucleases and sequencing, then RGN-EGFP were retransformed into pcDNA3.1 （ ＋ ）-zcocin by restriction endonucleases, the recombinant pcDNA3.1-RGN-EGFP-zeo was transformed into E. cob. DH5α,identified by double digestion with restriction endonudeases and sequencing.The sequence of RGN full-length cDNA was confinned by blasting to Genbank. The recombinant plasmids were transfected into HK-2 cells by hpofectamine method. The expression of RGN mRNA in HK-2 cells was assayed by fluorescence microscopy and laser scanning confocal mieroscopy（LSCM）.Results The sequence of RGN eDNA was corrected by blasting to Geubank. The RGN fusion proteins were expressed in the cells transfected with pcDNA3.1 -RGN-EGFP-zeo plasmid. Green fluorescence was observed by fluorescenece microscopy and LSCM on the cells transfeced with pcDNA3.1-RGN-EGFP-zeo plasmid. Condusion The recombinant eukaryotic expression vector pcDNA3.1-RGN-EGFP-zeo was suceesfully constructed and expressed in the HK-2 cells.