DCSIGN特异性适配子筛选及鉴定.docVIP

DCSIGN特异性适配子筛选及鉴定 　作者：陈芳, 曾洁, 孙平, 潘勤, 章晓联 【摘要】 　　目的: 筛选高特异性、 高亲合力结合DCSIGN的单链DNA适配子, 为开发抗HIV、 HCV和结核杆菌感染的新型预防和治疗试剂奠定基础。 方法: 采用基于细胞表面展示的SELEX技术（TECSSELEX）, 筛选出具有高亲合力结合DCSIGN的单链DNA（ssDNA）适配子; 运用流式细胞术（FCM）检测该适配子的亲合力; 对最高亲合力适配子库进行克隆和测序; ELISA和FCM方法检测单适配子ZD8与DCSIGN蛋白结合的剂量相关性; MTT法测定IC50观察适配子对细胞的毒性。 结果: 第14轮筛选的适配子库亲和力最高; DCSIGN抗体能抑制适配子结合表达DCSIGN蛋白的细胞; 第14轮库中筛选的单适配子ZD8 与DCSIGN蛋白的结合率呈剂量依赖性; 所筛选的单适配子对肝细胞几乎无毒性。结论: 成功筛选出DCSIGN蛋白的ssDNA适配子, 为防治HIV、 HCV和结核杆菌感染等DCSIGN相关性疾病, 提供新型预防和治疗的候选试剂和小分子药物。 　　AIM: To develop the high affinity DNA aptamers which can selectively recognize DCSIGN protein as putative preventive reagents and drugs against infectious diseases. METHODS: A technique of cell surfaceSELEX (TECSSELEX) was employed to screen the DNA aptamers recognizing DCSIGN displayed on the surface of NIH3T3 cells. Some aptamers from the aptamer pools with the highest affinity were cloned. The binding affinities between the aptamers and target cells were measured by flow cytometry and ELISA. The values of IC50 were determined to detect the toxicity of aptamers for cells by MTT method. RESULTS: The 14th aptamer pool had the highest affinity and the binding rates between single aptamer (ZD8) and DCSIGN protein were in a dose dependent manner. DCSIGN antibody can competitively inhibit the binding reactions between the aptamers and their target cells. The selected aptamers have very low or no toxicity for hepatic cells. CONCLUSION: The selected DNA aptamers bound to DCSIGN protein with high affinity and specificity have been successfully selected, and they can be used both as preventive reagents and drugs against infectious diseases, such as AIDS, hepatitis C and tuberculosis. 【关键词】 DCSIGN; 适配子; TECSSELEX 　　DCSIGN 可通过其对靶细胞的捕捉, 将病原体直接带到靶细胞附近, 增加感染机会, 使DC成为病原体的传播者。近来的研究证明, DCSIGN为广谱的病原受体, 除了可与HIV1、 HCV结合外, 还可与其他多种病原体结合［1］。因而, 通过阻断DCSIGN从而阻断HIV、 HCV、 结核杆菌等病原体感染途径有望成为预防和治疗病毒性疾病的有效策略。 　　SELEX技术是在体外运用大容量的随机寡核苷酸文库与PCR扩增技术相结合, 以指数富集与靶分子特异结合的寡核苷酸, 经过多轮筛选, 获得高亲合力、