小鼠CD160胞外段真核表达载体的构建和表达

Construction and Expression of Eukaryotic Expressing Vector of Mouse Soluble CD160

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 expressing vector of mouse soluble CD160 and stably transfect into CHO cells for eukaryotic expression.Methods Recombinant soluble CD160（rsCD160）was constructed by gene recombination.Total RNA was extracted from the spleen of C57BL/6 mice.cDNA was amplified for the soluble form of CD160.Then,the PCR product was cloned to pcDNA3.1 and pEGFP-N1.The recombinant plasmid was identified by restriction map and sequence analysis.The soluble CD160 expression in CHO cells transfected with recombinant psCD160 was verified by RT-PCR and Western blot.The binding ability of psCD160 to its ligand was detected by FACS.Results 520 bp mouse soluble CD160 gene was obtained.Recombinant mouse psCD160 was successfully constructed.After transfection,soluble CD160 expression in the culture supernatant of CHO cells was successfully detected.FACS analysis indicated that soluble CD160 could bind to its ligand.Conclusion Recombinant mouse psCD160 is successfully constructed,which多 will benefit our further study on soluble CD160 for immune therapy against tumor in the future experiments.