人OX40-Fc分子的构建、真核表达及活性研究

目的构建人OX40-Fc基因的真核表达质粒，并表达有生物学活性的纯化人OX40-Fc融合蛋白。方法从活化的人T细胞cDNA文库中以PCR方法扩增OX40膜外区cDNA编码基因，并导入真核表达载体pcDNA3中。测序证实后，进行酶切并与人IgG1Fc基因一起装入真核表达载体pcDNA3中，采用脂质体法稳定转染COS-7细胞，用夹心ELISA检测其表达情况，经蛋白A亲和纯化，用SDS-PAGE与Western blotting进行鉴定。检测其对活化后Jurkat细胞增殖的抑制活性。结果测序证实构建的OX40膜外区与OX40-Fc cDNA阅读框完整，连接部位序列正确；ELISA证实OX40-Fc蛋白的表达；SDS-PAGE与Western blotting证实其为OX40-Fc蛋白。增殖实验证明其对活化后的Jurkat细胞有抑制增殖作用。结论成功构建了人OX40-Fc真核表达载体，并使有生物学活性的OX40-Fc融合蛋白在COS-7细胞上获得了稳定表达。为进一步研究该蛋白在自身免疫性疾病治疗中的作用及调节免疫自稳机制中的地位奠定了基础。

Construction, eukaryotic expression, and characterization of human OX40-Fc molecule

Objective To construct eukaryotic expression plasmid of hOX40-Fc gene and stably express hOX40-Fc fusion protein with biological activity. Methods The extramembrane encoding region of OX40 molecule was cloned from a normal human activated T cells cDNA library by polymerase chain reaction. After sequencing, the extramembrane encoding region together with human IgG1-Fc cDNA was inserted into the eukaryotic expression plasmid pcDNA3. The right recombinant was transfected into COS-7 cells with lipofectamine reagent. OX40-Fc protein was purified by recombinated protein A affinity column chromatography. The molecular weight, purity, and antigenicity of OX40-Fc were identified by sandwich ELISA, SDS-PAGE, and Western blotting. Results The open-reading frame of OX40-Fc gene was coincident with what we had expected. OX40-Fc protein expression in COS-7 cells was confirmed, and the antigenicity of the purified OX40-Fc protein was analyzed. The purified OX40-Fc protein could inhibit the growth of activated Jurkat cells by proliferation assay. Conclusion The vector is constructed successfully and a purified recombinated OX40-Fc protein is obtained. This lays a foundation for further studies of OX40, such as its role in autoimmune diseases and immune homeostasis.