牛PU.1基因真核表达载体的构建、表达及鉴定

摘要

本研究从牛外周血单个核细胞提取细胞总RNA,RT-PCR获得编码牛PU.1蛋白的cDNA；以pIRES2 DsRed-Express2为骨架构建牛PU.1基因表达载体pIRES2 DsRed-Express2-PU.1,并利用脂质体介导重组质粒分别转染H293T细胞和牛胎儿成纤维细胞,通过荧光观察和Western blotting鉴定表明成功构建了牛PU.1的表达载体,为下一步研究牛血单核细胞分化及牛抗病育种奠定基础.%Total RNA was extracted from bovine,the cDNA encoding PU. 1 protein was cloned using reverse transcription polymerase chain reaction (RT-PCR). The PU. 1 gene was subcloned into pIRES2 DsRed-Express2 vector and then the recom-binant plasmids were transferred into H293T cells and bovine fetal fibroblasts cells by the method of lipofectaming. The results showed that we had successfully constructed the expression plasmid of PU. 1 gene by fluorescence observation and identification of Western blotting, which layed the foundation for the differentiation of bovine blood monocytes and breeding for disease resistance in cattle.