pIRES2-EGFP-hIL-10真核表达载体的构建及鉴定

Construction and Identification of pIRES2-EGFP-hIL-10 Plasmid

目的构建带有报告基因EGFP的hIL-10真核表达载体。方法通过RT-PCR方法从人外周血淋巴细胞中扩增出IL-10基因,构建IL-10基因和报告基因EGFP基因真核表达载体pIRES2-EGFP-hIL-10,经PCR、酶切、测序等方法进行鉴定,紫外分光光度计测定质粒浓度及纯度。结果酶切、PCR及测序证实pIRES2-EGFP-hIL-10载体序列正确。结论成功构建真核表达载体pIRES2-EGFP-hIL-10,可为后续器官移植免疫耐受的研究奠定基础。

Objective To construct the pIRES2-EGFP-hIL-10 plasmid of human. Methods IL-10 gene was amplified from human peripheral blood lymphocytes by using RT-PCR,and then pIRES2-EGFP-hIL-10 plasmid was constructed. The identification was done by using endonuclease eutting,PCR and sequencing and the plasmid concentration and purity were detected by using UV spectrophotometer. Results Endonuclease cutting. PCR and sequencing showed the successful construction of pIRES2-EGFP-hIL-10 plasmid. Conclusion We construct pIRES2-EGFP-hIL-10 plasmid successfully and lay the foundation for immunotolerance researches in the organ transplantation.