pEGFP-BLCAP真核表达载体的构建和鉴定

目的：构建真核表达载体pEGFP—BLCAP并转染骨肉瘤细胞SOSP—M。方法：从培养的人骨肉瘤细胞系SOSP—M细胞中提取总RNA，经RT—PCR获得BLCAP基因的cDNA，测序正确后插入真核表达载体pEGFP—C2中。构建的重组质粒经脂质体介导转染SOSP—M细胞，经观察荧光蛋白表达和Western blot鉴定目的蛋白在转染后的SOSP—M细胞中的表达情况。结果：RT—PCR成功地扩增出一条约280bp的片段，经限制性内切酶分析和DNA序列测定证实目的基因cDNA已插入重组质粒；荧光显微镜下观察到转染后的SOSP—M细胞发出较强绿色荧光，Western blot证明BLCAP能以融合蛋白的形式在SOSP—M细胞中获得表达。结论：构建了真核表达载体pEGFP—BLCAP并成功表达目的蛋白。

Construction and identification of eukaryotic expression vector pEGFP-BLCAP

Objective:To construct eukaryotic expression vector pEGFP-BLCAP and transfected into SOSP-M osteosarcoma cells.Methods:From cultured human osteosarcoma SOSP-M cell line,total RNA was extracted.BLCAP gene was amplified by RT-PCR and inserted to the eukaryotic expression vector pEGFP-C2.The recombinant plasmid was transfected into SOSP-M cells by liposome.The expression of EGFP-BLCAP was identified by Western blot and observing green fluorescence.Results:A 280bp specific fragment was amplified.Restriction enzyme analysis and DNA sequencing confirmed that the gene has been inserted into recombinant plasmid;green fluorescence could be observed after transfection;western blot analysis demonstrated that the fusion protein EGFP-BLCAP was able to express in transfected SOSP-M cells.Conclusion:Construction of the eukaryotic expression vector is successful.