CAJ | 학술논문

目的：制备抗人 OX40单克隆抗体，为进一步研究OX40/OX40L 通路以及针对该通路进行疾病干预、疾病治疗和药物筛选奠定基础。方法 PCR扩增人 OX40胞外区的基因序列，将其构建到原核表达载体 pET-32a(+)中，利用大肠杆菌表达系统表达OX40胞外区蛋白，经 Ni 柱纯化后获得目的蛋白。以纯化的 OX40胞外区蛋白为免疫原免疫小鼠，采用常规方法进行细胞融合，通过 ELISA 和多次克隆化培养，筛选出分泌特异性鼠抗人 OX40单克隆抗体的杂交瘤细胞株；通过ELISA、Western blot 和免疫荧光检测抗体的效价及特异性。结果成功构建人 OX40原核表达质粒 pET-32a(+)-OX40，并在 BL21(DE3)中诱导表达，经 SDS-PAGE 和 Western blot 鉴定、Ni 柱纯化、透析后获得 OX40胞外区蛋白；以此纯化蛋白为免疫原，成功制备具有较强亲和力的单克隆抗体。结论克隆表达并纯化了人 OX40蛋白，成功制备针对该蛋白的高亲和力单克隆抗体，为进一步研究 OX40/OX40L的生物学功能奠定了实验基础。
목적：제비항인 OX40단극륭항체，위진일보연구OX40/OX40L 통로이급침대해통로진행질병간예、질병치료화약물사선전정기출。방법 PCR확증인 OX40포외구적기인서렬，장기구건도원핵표체재체 pET-32a(+)중，이용대장간균표체계통표체OX40포외구단백，경 Ni 주순화후획득목적단백。이순화적 OX40포외구단백위면역원면역소서，채용상규방법진행세포융합，통과 ELISA 화다차극륭화배양，사선출분비특이성서항인 OX40단극륭항체적잡교류세포주；통과ELISA、Western blot 화면역형광검측항체적효개급특이성。결과성공구건인 OX40원핵표체질립 pET-32a(+)-OX40，병재 BL21(DE3)중유도표체，경 SDS-PAGE 화 Western blot 감정、Ni 주순화、투석후획득 OX40포외구단백；이차순화단백위면역원，성공제비구유교강친화력적단극륭항체。결론극륭표체병순화료인 OX40단백，성공제비침대해단백적고친화력단극륭항체，위진일보연구 OX40/OX40L적생물학공능전정료실험기출。
Objective To prepare novel anti-OX40 functional monoclonal antibodies and characterize their distinct biological functions. Methods The gene fragment of extracellular region of OX40 was amplified by PCR and cloned into pET-32a(+) prokaryotic expressing vector. The expressed products were purified by Ni sepharose and identified by Western blot. The purified recombinant protein was used as antigen to immunize BALB/c mice. Then the immunized spleen cells were isolated from immunized mice and fuse with Sp2/0, a kind of myloma. After screening by ELISA, hybridomas secreting anti-OX40 mAb were acquired and used to generate specific Abs. At last, biological activities of Abs were investigated by ELISA, Western blot. Results The recombinant OX40 extracellular domain protein was successfully expressed in BL21 and purified by Ni sepharose. It was a protein with about 38 kD molecular weight as analyzed by SDS-PAGE and Western blot. The data of FACS demonstrated that the antiserum had high affinity to OX40 expressed on the membrane of OX40-transfected cells. Conclusion The purified protein OX40 has been successfully obtained. The prepared monoclonal antibody shows high specificity and titer in immunized mice. The preparation of recombinant OX40 and its monoclonal antibody has provided reliable tools for the future study on the biologic activity of OX40/OX40L.