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目的构建小鼠CD160胞外段的真核表达载体,并稳定转染CHO细胞进行真核表达。方法从C57BL/6小鼠脾脏中提取总RNA,经RT-PCR扩增CD160胞外段,然后将其克隆到真核表达载体pcDNA3.1和pEGFP-N1两种质粒中,构建重组质粒psCD160和pEGFP-sCD160。重组质粒经双酶切鉴定及测序正确后,用脂质体转染CHO细胞,并用RT-PCR和Western blot检测CD160胞外段的表达,流式细胞术检测重组psCD160与其配体的结合能力。结果获得长度约为520 bp的小鼠CD160胞外段基因,经测序证实其序列正确。用脂质体将含有CD160胞外段基因的重组质粒载体转染CHO细胞后,RT-PCR和Western blot分析证实转染的CHO细胞可表达重组的psCD160基因,并且表达的可溶性CD160可与其配体结合。结论成功构建小鼠CD160胞外段基因的真核表达载体,并转染CHO细胞后表达出相应蛋白,为进一步研究CD160胞外段的功能效应奠定实验基础。
Objective To construct eukaryotic expression vector of murine CD160 extracellular domain and stably transfected CHO cells for eukaryotic expression. Methods Total RNA was extracted from the spleens of C57BL / 6 mice. The extracellular domain of CD160 was amplified by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1 and pEGFP-N1. The recombinant plasmid psCD160 and pEGFP-sCD160. The recombinant plasmids were identified by double enzyme digestion and sequenced correctly. The recombinant plasmids were transfected into CHO cells by liposome. The expression of extracellular domain of CD160 was detected by RT-PCR and Western blot. The binding ability of recombinant psCD160 to its ligand was detected by flow cytometry. Results The extracellular domain of mouse CD160 gene, about 520 bp in length, was obtained and its sequence was verified by sequencing. RT-PCR and Western blot analysis confirmed that transfected CHO cells could express recombinant psCD160 gene after transfection of recombinant plasmid vectors containing CD160 extracellular domain gene with liposomes, and expressed soluble CD160 can bind with its ligand Combined. Conclusion The eukaryotic expression vector of mouse CD160 extracellular domain gene was successfully constructed and transfected into CHO cells to express the corresponding protein, which laid the experimental foundation for further study on the functional effect of CD160 extracellular domain.