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纸质出版:2008
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谢琳,刘韧,朱旭东,贺翔鸽,陈彩宇.人重组TGF-β RⅡ亲和核酸筛选方法的建立[J].陆军军医大学学报(原第三军医大学学报),2008,30(23):2168-2171.
XIE Lin,LIU Ren,ZHU Xu-dong,HE Xiang-ge,CHEN Cai-yu.Screening of nucleic acids affinity to recombinant human TGF-β RⅡ[J].J Amry Med Univ (J Third Mil Med Univ),2008,30(23):2168-2171.
目的 通过筛选转化生长因子-β受体Ⅱ(TGF-β RⅡ)亲和核酸,探寻拮抗TGF-β活性的新方法。 方法 以ssDNA 5′-TAATACGACTCACTATAGGGAGGACGATGCGG-N60-CAGACGACTCGCCCGA-3′为模板,体外转录获取初始RNA随机库;以人体外重组TGF-β RⅡ为靶目标蛋白行SELEX实验,共进行8轮循环筛选。通过膜结合实验监测筛选所得RNA富集库对TGF-β RⅡ亲和性的进化状态,凝胶阻滞实验检测亲和核酸序列与TGF-β RⅡ的结合强度。 结果 随着筛选进行,RNA富集库沿着与靶蛋白TGF-β RⅡ亲和性加强的方向进化;从第8轮富集库中分离出两类优势序列,序列A和序列B。膜结合实验显示序列A、B对TGF-β RⅡ有明显的亲和性,但凝胶阻滞实验显示序列A、B均无与蛋白结合的阻滞带出现。 结论 筛选所得序列A及B 与靶蛋白TGF-β RⅡ的亲和性及特异性还不理想,但先导序列A及B的获得为通过后SELEX技术进一步寻找最佳结合序列奠定了基础。
Objective To select the aptamer to an extracellular soluble fragment of recombinant human TGF-β receptor Ⅱ (TGF-β RⅡ) in order to antagonize TGF-β effectively by using systematic evolution of ligants by exponential enrichment (SELEX). Methods Initial random RNA library was transcripted in vitro from ssDNA 5′-TAATACGACTCACTATAGGGAGGACGATGCGG-N60-CAGACGACTCCCCGA-3′; rhTGF-β RⅡ was used as target protein. Totally
selection of 8 times was carried out in SELEX experiment. Membrane binding assay was performed to detect the evolution of enriched RNA library; Electrophoretic mobility shift assay (EMSA) was done to determine the affinity between the selected nucleic acid sequence and TGF-β RⅡ. Results Evolution of the enriched RNA library along the increased affinity to TGF-β RⅡ was observed with the development of selection. Two types of dominant sequences were isolated and named as sequence A and sequence B. In membrane binding assay
both sequences A and B showed obvious affinity to TGF-β RⅡ. However
no retarded bands were seen in EMSA. Conclusion The affinity of sequences A and B to TGF-β RⅡ is beyond satisfaction. However
possible sequences with improved affinity to TGF-β RⅡ can be selected by post-SELEX on the basis of candidate sequences A and B.
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