小鼠CD25分子胞外段的真核表达载体的构建

Construction and identification of eukaryotic expression vector of mouse CD25 extracellular domain

目的构建小鼠CD25分子胞外段的真核表达载体,并在CHO细胞中进行表达。方法从前期构建的原核表达载体PET-32a-CD25胞外段上酶切下CD25胞外段;将其亚克隆入真核表达载体pcDNA3.1,并进行酶切及测序鉴定;用Lipofectamine 2000将pcDNA3.1-CD25胞外段转染中国仓鼠卵巢细胞(Chinese Hamster Ovary,CHO)进行表达,并用ELISA法对表达产物进行测定。结果酶切、测序验证成功构建真核表达载体pcDNA3.1-CD25e,并在CHO细胞中有效表达CD25e蛋白。结论成功构建小鼠CD25胞外段的真核表达载体,可在CHO细胞中表达,并具有良好的免疫反应性,为后续实验提供了前期基础。

Objective To construct a eukaryotic expression vector of mouse CD25 extracellular domain and to express it in CHO. Methods The prokaryotie expression vector PET-32a-CD25e was digested with EcoR I and Hind III. The target sequence was subeloned into eukaryotic expression vector pcDNA3.1/Myc-his- （-）. The recombinant plasmid was identified through enzyme cleaving and sequencing before expression. The recombinant plasmids were transferred into ClIO by Lipofectamine 2000. The concentration of target molecule in supematant was detected by ELISA assay. Results The recombinant plasmid pcDNA3.1-CD25 extracellular domain gene was identified by sequencing and could express CD25e protein in CHO cells. Conclusions The mouse CD25 extracellular domain has been successfully expressed in eukaryotic cells, which provided the foundation for further relative studies.