mOX40-Fc的真核表达及其对混合淋巴细胞反应的影响

构建重组pcDNA3.1-mOX40-Fc真核表达载体,在CHO细胞中表达,并初步研究融合蛋白mOX40-Fc对MLR中细胞增殖的影响。克隆编码hIgG1Fc基因片段,与编码小鼠OX40胞外段的基因融合,构建mOX40-Fc融合基因及其分泌型真核表达载体pcDNA3.1-mOX40-Fc;转染CHO细胞构建融合蛋白的稳定真核表达细胞株,用RT-PCR及夹心ELISA检测其表达,纯化后经SDS-PAGE及Western blot法对其进行鉴定,混合淋巴细胞反应(MLR)测定其生物学活性。结果:核苷酸序列测定显示构建的mOX40胞外段和hIgG1Fc段基因序列与GenBank序列一致、阅读框完整;RT-PCR、ELISA和Western blot检测表明转染细胞有分泌型重组蛋白的表达;功能实验提示mOX40-Fc能抑制MLR中的细胞增殖。成功构建mOX40-Fc真核表达载体并获得高效表达,该重组蛋白可在体外通过抑制OX40/OX40L共刺激通路抑制淋巴细胞增殖。

Eukaryotic expression of the secretory mOX40-Fc fusion protein and its effects on mixed lymphocyte reaction

To construct a recombinant eukaryotic expression vector containing pcDNA3.1-mOX40-Fc fusion gene,to express functional fusion protein in the CHO cells and to study the effects of mOX40-Fc on MLR,cDNA encoding the hIgG1Fc was prepared by RT-PCR from the RNA of the stimulated human peripherial blood lymphocyte,and mOX40 extracellular gene was cloned from pIRES2-EGFP-OX40 by PCR.Two amplified PCR products were ligated into a secrectory mammalian expression vector pcDNATM3.1/myc-HisA.The recombined vector was transfected into CHO cells and its expression was detected by RT-PCR and sandwich ELISA.After purified by protein A affinity column chromatography,the mOX40-Fc fusion protein was identified by SDS-PAGE and Western blotting.And then,its effect of on cell proliferation in MLR was investigated by 3H-TdR method.The result showed that the ORF of mOX40-Fc gene was coincident with what we had expected the protein was correctly expressed and could inhibit cell proliferation in MLR.It is evident that the secretory mOX40-Fc fusion protein plays a inhibiting role in MLR.