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目的制备IL-12锚定修饰exosomes(EXOs)的肾癌疫苗及其蛋白组成和功能鉴定。方法将糖基化磷脂酰肌醇(Glycosylphosphatidylinositol,GPI)信号肽序列与白介素12基因融合构建真核双表达质粒pBIG。激光共聚焦显微镜和流式细胞仪检测融合蛋白(CPI-IL-12)在肾癌细胞的表达。用超滤和蔗糖重水密度梯度离心法制备EXOs,透射电镜鉴定其形态,Westernblot鉴定其标志性分子热休克蛋白70(HSP70)和细胞间粘附分子(ICAM-1)及肾癌特异性抗原G250和GPI-IL-12的表达。ELISA测定EXOs负载IL-12的量,IFN-γ释放试验检测IL-12修饰的EXOs(EXO/IL-12)的功能。结果真核双表达质粒pBIG构建成功,酶切鉴定及测序正确。稳定转染后,GPI-IL-12在肾癌细胞膜高表达。分离纯化的EXOs为类圆碟形、双层膜结构,直径30～80 nm,表达HSP70、ICAM-1、G250和GPI-IL-12。ELISA测定10μg/ml的EXOs含约(80±9.6)pg/ml的IL-12,并能显著诱导T淋巴细胞产生IFN-γ。结论pBIG质粒稳定转染肾癌细胞能制备EXO/IL-12疫苗。该疫苗表达GPI-IL-12,负载肾癌特异性抗原G250,能在体外显著诱导T淋巴细胞分泌IFN-γ,可望成为治疗肾癌的新疫苗。
OBJECTIVE: To prepare the renal carcinoma vaccine with IL-12 anchored exosomes (EXOs) and its protein composition and function identification. Methods Glycosylphosphatidylinositol (GPI) signal peptide sequence was fused with interleukin 12 gene to construct eukaryotic double expression plasmid pBIG. The expression of fusion protein (CPI-IL-12) in renal cell carcinoma was detected by confocal laser scanning microscopy and flow cytometry. EXOs were prepared by ultrafiltration and sucrose-dextrose gradient centrifugation. The morphology of the cells was identified by transmission electron microscopy. Western blot was used to identify the expression of HSP70, ICAM-1 and G250 And GPI-IL-12 expression. The amount of IL-12 loaded by EXOs was measured by ELISA, and the function of IL-12-modified EXOs (EXO / IL-12) was examined by IFN-γ release assay. Results The eukaryotic double-expression plasmid pBIG was successfully constructed, identified by restriction enzyme and sequenced correctly. After stable transfection, GPI-IL-12 is highly expressed in the kidney cancer cell membrane. The isolated and purified EXOs are disk-shaped and bilayer membranes with 30-80 nm in diameter and express HSP70, ICAM-1, G250 and GPI-IL-12. The 10 μg / ml of EXOs contained about 80 ± 9.6 pg / ml of IL-12 by ELISA and significantly induced T-lymphocyte production of IFN-γ. Conclusion EXO / IL-12 vaccine can be prepared by stably transfecting pBIG plasmid into renal cancer cells. The vaccine expresses GPI-IL-12 and supports the specific antigen G250 of kidney cancer, which can induce T lymphocyte to secrete IFN-γ significantly in vitro, which is expected to become a new vaccine for treating renal cell carcinoma.