CAJ | 학술논문

目的建立体外获得突变型p53蛋白DNA适配子(aptamers)的方法,为该适配子在肿瘤早期诊断及靶向治疗运用中奠定基础.方法构建长度为75 nt的初始随机单链DNA库,其两端为固定序列,中间为35个随机序列,库容量为1 × 1017～1×1018,以溴化氰活化的琼脂糖小球为筛选介质,运用指数富集的配体系统进化技术(SELEX技术)经逐轮筛选获得突变型p53蛋白的DNA适配子.将适配子库克隆、测序,采用DNAMAN软件对其结构进行分析,通过生物素-亲和素-辣根过氧化物酶显色系统测定适配子与突变型p53蛋白的亲和力.结果经逐轮筛选,所得适配子富集库与突变型p53蛋白的亲和力逐步提高,A450值从0.1357增加到了0.6818,其亲和力至第8轮筛选达最高水平后进入平台期.克隆第8轮筛选适配子库,测定23个克隆子的序列,其中20个克隆子的序列与预期相符.DNAMAN5.29软件分析表明,20个克隆子的一级序列同源率为76.51%,无共同保守序列,适配子的二级结构则以茎环、凸环结构为主.结论经8轮筛选获得的DNA适配子与突变型p53蛋白的体外结合具有高度的特异性及亲和力,其克隆子无共同保守序列,适配子的高亲和性与其形成的茎环、凸环等空间结构密切相关.
목적 건입체외획득돌변형p53단백DNA괄배자(aptamers)적방법,위해괄배자재종류조기진단급파향치료운용중전정기출.방법 구건장도위75 nt적초시수궤단련DNA고,기량단위고정서렬,중간위35개수궤서렬,고용량위1 × 1017～1×1018,이추화청활화적경지당소구위사선개질,운용지수부집적배체계통진화기술(SELEX기술)경축륜사선획득돌변형p53단백적DNA괄배자.장괄배자고극륭、측서,채용DNAMAN연건대기결구진행분석,통과생물소-친화소-랄근과양화물매현색계통측정괄배자여돌변형p53단백적친화력.결과 경축륜사선,소득괄배자부집고여돌변형p53단백적친화력축보제고,A450치종0.1357증가도료0.6818,기친화력지제8륜사선체최고수평후진입평태기.극륭제8륜사선괄배자고,측정23개극륭자적서렬,기중20개극륭자적서렬여예기상부.DNAMAN5.29연건분석표명,20개극륭자적일급서렬동원솔위76.51%,무공동보수서렬,괄배자적이급결구칙이경배、철배결구위주.결론 경8륜사선획득적DNA괄배자여돌변형p53단백적체외결합구유고도적특이성급친화력,기극륭자무공동보수서렬,괄배자적고친화성여기형성적경배、철배등공간결구밀절상관.
Objective To set up a method to obtain single-stranded DNA aptamers of mutant p53 protein in vitro by SELEX technique and lay a foundation of application of these aptamers to carcinoma in early diagnosis and targeted therapy. Methods The initial length of 75 bp random single-stranded DNA library was constructedin vitro, both ends had a fixed sequence and there were 35 nucleotides in the middle of random sequence, with capacities of about 1×1017-1×1018. By means of cyanogen bromide (CNBr)activated agarose beads as screening medium, the ssDNA aptamers of mutant p53 protein were selected by SELEX technique. The aptamers were cloned and sequenced, and DNAMAN package was employed to analyze the conserved equences and the second structure of the aptamers. The affinity of aptamers to mutant p53 protein was determined by chromatic biotin-streptavidin-horseradish peroxides system. Results Through screening aptamers round by round, the affinity of aptamers to mutant p53 protein was increased gradually(the A450 values were increased from 0.1357 to 0.6818) and the affinity of aptamers at the round 8 was the highest, and then maintained at the prateau. Twenty out of 23 aptamers had the correct length and fixed sequence after cloning and sequencing. The DNAMAN5.29 sofeware analysis revealed that, the identity of 20 clones was 76. 51% without common conserved sequence and the second structure of the aptamers was stem-loop-based and bulge-based. Conclusion After selection of 8 rounds, the ssDNA aptamers we obtained had high specificity and affinity to mutant p53 protein in vitro and didn't have common conserved sequence. Stem-loops and bulge were the basis of aptamers binding to mutant p53 protein.