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目的:制备抗人骨桥蛋白(hOPN)的单克隆抗体(mAb),用于其功能研究。方法:构建OPN的原核表达载体pTriEx-1-hONP载体,转化Tuner(DE3)placI感受态大肠杆菌,用IPTG进行诱导表达。利用镍柱亲和层析纯化OPN蛋白,纯化蛋白经超滤浓缩洗涤后即为免疫原。以重组纯化的OPN蛋白为免疫原,免疫BALB/c小鼠,取免疫小鼠的脾细胞和同系小鼠的骨髓瘤细胞Sp2/0进行常规融合,通过有限稀释法进行克隆和间接ELISA筛选,获得分泌鼠抗人OPN蛋白mAb的杂交瘤细胞株,通过ELISA、Western blot等方法检测其特异性,并用免疫组化方法检测了正常月经周期子宫内膜OPN的表达。结果:pTriEx-1-hONP表达的OPN蛋白主要为可溶性形式,经镍柱纯化后,蛋白纯度可达85%以上。纯化的OPN重组蛋白免疫小鼠后经融合筛选,得到2株稳定分泌抗人OPN的mAb杂交瘤细胞株,分别命名为1E7和5B7,两株mAb的免疫球蛋白亚类分别IgG1和IgG2a。通过ELISA和Western blot等方法鉴定,抗OPN的mAb能与OPN蛋白特异性结合。通过免疫组织化学方法对不同时期的正常子宫内膜的OPN的检测表明,子宫内膜腺上皮在增生期、分泌早期,OPN呈弱阳性表达;分泌中、晚期OPN呈强阳性表达。结论:以纯化的重组hOPN为免疫原成功制备了鼠抗hOPN的mAb,并初步进行了应用,为研究hOPN的功能打下了良好基础。
OBJECTIVE: To prepare a monoclonal antibody (mAb) against human osteopontin (hOPN) for its functional study. Methods: The prokaryotic expression vector pTriEx-1-hONP was constructed and transformed into Tuner (DE3) placI competent E.coli and induced by IPTG. OPN protein was purified by nickel column affinity chromatography and the purified protein was concentrated by washing with ultrafiltration to be the immunogen. BALB / c mice were immunized with the purified recombinant OPN protein. The splenocytes of immunized mice and Sp2 / 0 of myeloma cells of the same line mice were routinely fused. The cloned and indirect ELISA were screened by limiting dilution method. The hybridoma cell line secreting mouse anti-human OPN protein mAb was obtained. The specificity of the hybridoma cell line was detected by ELISA and Western blot. The expression of OPN in normal menstrual cycle was detected by immunohistochemistry. Results: The OPN protein expressed by pTriEx-1-hONP was mainly in soluble form. After purified by nickel column, the purity of protein reached more than 85%. The purified OPN recombinant protein was immunized in mice and then screened by fusion. Two mAb hybridoma cell lines stably secreting anti-human OPN were obtained and named as 1E7 and 5B7 respectively. The immunoglobulin subclasses of the two mAbs were IgG1 and IgG2a, respectively. Anti-OPN mAbs could specifically bind to OPN protein by ELISA and Western blot. Immunohistochemical staining of OPN in different stages of normal endometrium showed that the expression of OPN was weakly positive in the proliferative and early stages of endometrial glandular epithelial cells. CONCLUSION: The mouse anti-hOPN mAb was successfully prepared using the purified recombinant hOPN as the immunogen. The mAb was initially applied and laid a good foundation for the study of the function of hOPN.