6B11ScFv-mHSP70融合蛋白诱导抗卵巢癌免疫应答的体外实验研究

Anti-ovarian tumor immune responses induced by the fusion protein of anti-idiotypic single chain antibody-heat shock 70 in vitro

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 Anti- idiotypic single chain antibody 6BllScFv can mimic OC166 - 9, the antigen of ovarian carcinoma in our previous research. This study is aimed to evaluate the immunogenesis of the fusion protein, 6BllScFv - mHSP70 in vitro; and to explore the probability of 6BllScFv- mHSP70 as an ovarian cancer vaccine. Methods 6BllScFv-mHSP70 was expressed by E. coil and the fusion protein was higher than 95% purity after denature and renaturation. The immune activity of 6BllScFv-mHSP70 was analyzed with ELISA. Isolated female peripheral blood mononuclear cells （PBMC） expressing HLA- A2 were cocultured and stimulated with 6BllScFv-mHSP70. The proliferations of PBMC and cytotoxicity were examined by CCK-8 and LDH release test respectively. Phenotype of T lymphocyte stimulated by 6BllScFv-mHSP70 was analyzed by immune flow cytometry. Resuits 6BllScFv-mHSP70 showed capacity of binding to ovarian cancer monoclonal antibody COC166-9 and anti-mHSP70 antibody. It also could stimulate PBMC to proliferate and presented cytotoxicity to ovarian carcinoma cells expressing 012166 -9 and HLA-A2. Compare to untreated cells, 6BllScFv- mHSP70 treated lymph cells showed phenotypes as significantly increased in CD4＋T cell, slightly increased in CD8 ＋T cell, significantly increase in CD4＋/CD8＋ ratio and significantly decreased in （CD4＋CD25＋） /CD4＋ ratio. Conclusions 6BllScFv - mH-SP70 can induce cellular immunity against ovarian carcinoma in vitro. This finding provides experimental evidence for clinical use of 6B11ScFv - mHSP70 as anti - idiotype vaccine against ovarian carcinoma.