| IL-12锚定修饰exosome的肾癌疫苗的制备及鉴定 |
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| 引用本文： | 张尧,吴小侯,陈刚,罗春丽,张家摸.IL-12锚定修饰exosome的肾癌疫苗的制备及鉴定[J].免疫学杂志,2010(4). |
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| 作者姓名： | 张尧 吴小侯 陈刚 罗春丽 张家摸 |
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| 作者单位： | 重庆医科大学附属第一医院泌尿外科;重庆医科大学检验教研室; |
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| 摘 要： | 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...
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| 关 键 词： | 肾细胞癌 Exosomes 白细胞介素12 肿瘤疫苗 |
Preparation and identification of the renal-cancer vaccine of exosome modified to express GPI-anchored IL-12 |
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| ZHANG Yao,WU Xiaohou,CHEN Gang,LUO Chunli,ZHANG Jiamo.Preparation and identification of the renal-cancer vaccine of exosome modified to express GPI-anchored IL-12[J].Immunological Journal,2010(4). |
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| Authors: | ZHANG Yao WU Xiaohou CHEN Gang LUO Chunli ZHANG Jiamo |
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| Institution: | ZHANG Yao,WU Xiaohou,CHEN Gang,LUO Chunli,ZHANG Jiamo Department of Urology,First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China |
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| Abstract: | 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 ... |
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| Keywords: | Renal cell carcinoma Exosome Interleukin 12 Tumor vaccine |
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