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目的既往研究证实,6B11抗独特型单链抗体(6B11ScFv)具有模拟卵巢癌相关抗原OC166-9的作用。本文研究6B11ScFv与小鼠热休克蛋白70(mHSP70)构建而成的融合蛋白(6B11ScFv-mHSP70)在体外抗卵巢癌免疫应答,探讨其作为卵巢癌疫苗的可能性。方法以大肠杆菌表达6B11ScFv-mHSP70融合蛋白,经变性复性后纯度达95%。采用ELISA检测其免疫学活性。采集HLA-A2阳性的女性健康志愿者的外周血单个核细胞,用6B11ScFv-mHSP70刺激并培养。采用CCK-8法、LDH释放法分别观察淋巴细胞增殖情况和对卵巢癌细胞系的细胞毒作用,流式细胞术检测6B11ScFv-mHSP70刺激前后淋巴细胞表型的改变。结果6B11ScFv-mHSP70具有特异性结合卵巢癌单抗COC166-9及抗小鼠-HSP70抗体的双重免疫学活性;可以刺激人外周血淋巴细胞的增殖;并对HLA-A2阳性、OC166-9表达阳性的卵巢癌细胞系有细胞毒作用;经6B11ScFv-mHSP70刺激后,实验组的淋巴细胞表型与未经蛋白刺激的对照组相比:CD4+T细胞明显增加,CD8+T细胞略有增加。CD4+/CD8+的比率明显增加,CD4+CD25+T细胞占CD4+T细胞的比率明显下降,差异有统计学意义。结论6B11ScFv-mHSP70融合蛋白在体外可诱导特异性抗卵巢癌的细胞免疫反应,有可能作为抗独特型疫苗用于卵巢癌的主动免疫。
Objective Previous studies have confirmed that the 6B11 anti-idiotypic single chain antibody (6B11ScFv) has the potential to mimic the ovarian cancer-associated antigen OC166-9. In this paper, we investigated the anti-ovarian cancer immune response of 6B11ScFv and mouse heat shock protein 70 (mHSP70) fusion protein (6B11ScFv-mHSP70) in vitro to explore its potential as a vaccine for ovarian cancer. Methods The 6B11ScFv-mHSP70 fusion protein was expressed in Escherichia coli and its purity was 95% after denaturation. The immunological activity was detected by ELISA. Peripheral blood mononuclear cells of HLA-A2-positive female volunteers were collected and stimulated with 6B11ScFv-mHSP70 and cultured. CCK-8 method and LDH release method were used to observe the proliferation of lymphocytes and the cytotoxic effect on ovarian cancer cell lines respectively. The changes of lymphocyte phenotypes were detected by flow cytometry before and after stimulation with 6B11ScFv-mHSP70. RESULTS: 6B11ScFv-mHSP70 had dual immunological activity that specifically binds to COC166-9 and anti-mouse-HSP70 antibodies in ovarian cancer cells. It stimulated the proliferation of human peripheral blood lymphocytes and expressed HLA-A2 positive and OC166-9 Of ovarian cancer cell lines had cytotoxicity. After stimulation with 6B11ScFv-mHSP70, the lymphocyte phenotype of the experimental group was significantly increased compared with the control group without protein stimulation: CD8 + T cells increased slightly. The ratio of CD4 + / CD8 + increased significantly, and the ratio of CD4 + CD25 + T cells to CD4 + T cells decreased significantly. The difference was statistically significant. Conclusion The 6B11ScFv-mHSP70 fusion protein can induce specific cellular immune responses against ovarian cancer in vitro and may be used as an anti-idiotypic vaccine in the active immunization of ovarian cancer.