共刺激分子配体B7-H1基因真核表达载体和转基因细胞的构建

The constructs of eukaryotic expression vector and transgenic cell of B7-H1

以PHA刺激的小鼠外周血单核细胞中抽提的总RNA为模板,通过RT-PCR技术,扩增出B 7-H 1基因。按常规方法克隆进pEF 6V 5H is A载体,重组质粒经鉴定后采用脂质体法转染CHO细胞,48 h后进行间接免疫荧光试验,72 h后用B lastic id in进行筛选,挑选出能稳定表达B 7-H 1的细胞株。结果表明:成功地克隆小鼠B 7-H 1基因并构建了用于表达B 7-H 1基因的真核表达载体,经间接免疫荧光试验证明,该基因在CHO细胞中得到表达。经W estern-b lotting检测表明筛选出的转基因细胞稳定表达了B 7-H 1基因。

Total RNA was isolated from mouse peripheral blood mononuclear cells and sub）ected to reverse transcription, and then the mouse BT-H1 gene was amplified by RT-PCR and then was cloned into the eukaryotic expression vector pEF6/VSHis A vector. The recombinant plasmids identified by restriction endonuclease analysis were transfected into CHO cells with liposome transfection reagent for transient expression. The target protein was successfully detected by IFA and western-blot.