IL-12锚定修饰exosome的肾癌疫苗的制备及鉴定

Preparation and identification of the renal-cancer vaccine of exosome modified to express GPI-anchored IL-12

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。

In the study,we aimed to prepare IL-12-anchored exosomes derived from renal cancer cells and identify their structure and function.Firstly,we constructed a mammalian co-expression plasmid of GPI-anchor-IL-12 by cloning fusion gene of GPI-anchor signal sequence and p40 cDNA in pBudCE4.1.Then we analyzed the expression of fusion protein by using confocal laser scanning microscopy and flow cytometry,and confirmed the IL-12 expression on the cell surface.Ultrafiltration and sucrose gradient centrifugation were employed to purify the exosomes,then TEM and Western blotting showed the exosomes were 30-80 nm in diameter with typical saucer-shape morphology,and expressing HSP70,ICAM-1,G250 and GPI-IL-12.About（80±9.6） pg/ml of IL-12 was detected in 10μg/ml exosome which could significantly induce the release of IFN-γ.The results suggest that the vaccine of exosomes-GPI -IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12,which expressing IL-12 and tumor associated antigen G250,and might be a new strategy for treatment of renal cancer.