CAJ | 학술논문

目的：筛选、鉴定人慢性粒细胞白血病融合蛋白BCR－ABL适配子。方法：利用SELEX（ Systematic evolution of ligands by exponential）技术，以高纯度融合蛋白BCR－ABL为靶分子，从体外化学合成的长度为90 bp的随机单链DNA文库中来筛选与融合蛋白BCR－ABL特异性结合的寡核苷酸适配子，并进行解离常数（ Kd）值测定和适配子序列测定，再分别用Clustal W软件包和DNA Folding Sever分析适配子一级结构及二级结构，以酶联仪测定OD450值，根据OD值高低判定适配子亲和力大小。结果：经过13轮筛选，随机ssDNA文库与融合蛋白的亲和率从0．3％上升到47．1％，所有的一级结构没有共同的同源序列，二级结构分析结果显示，茎和环等二级结构可能是适配子和融合蛋白BCR－ABL结合的基础，其中，寡核苷酸适配子A2与BCR－ABL亲和力最高，kd值达72 nmol／L。结论：利用随机寡核苷酸文库筛选技术成功获得抗融合蛋白适配子，为临床上治疗和预防慢性粒细胞白血病提供一定的参考。
목적：사선、감정인만성립세포백혈병융합단백BCR－ABL괄배자。방법：이용SELEX（ Systematic evolution of ligands by exponential）기술，이고순도융합단백BCR－ABL위파분자，종체외화학합성적장도위90 bp적수궤단련DNA문고중래사선여융합단백BCR－ABL특이성결합적과핵감산괄배자，병진행해리상수（ Kd）치측정화괄배자서렬측정，재분별용Clustal W연건포화DNA Folding Sever분석괄배자일급결구급이급결구，이매련의측정OD450치，근거OD치고저판정괄배자친화력대소。결과：경과13륜사선，수궤ssDNA문고여융합단백적친화솔종0．3％상승도47．1％，소유적일급결구몰유공동적동원서렬，이급결구분석결과현시，경화배등이급결구가능시괄배자화융합단백BCR－ABL결합적기출，기중，과핵감산괄배자A2여BCR－ABL친화력최고，kd치체72 nmol／L。결론：이용수궤과핵감산문고사선기술성공획득항융합단백괄배자，위림상상치료화예방만성립세포백혈병제공일정적삼고。
Objective:To screen and characterize aptamers against BCR-ABL fusion protein.Methods:A 90bp single stranded DNA( ssDNA) random library was subjected to 13 rounds of selection against BCR-ABL fusion protein by systematic evolution of ligands by expotential enrichment ( SELEX ) method, the selected aptamers were cloned and sequenced.The primary sequences and structure of aptamers were analyzed by Clustal W and DNA Folding Sever and the percentage of the ssDNA pool bound to BCR-ABL core protein were determinated.Results: after 13 rounds selection, the percentage of ssDNA pool bound to BCR-ABL fusion protein increased from 0.3%to 47.1%,the results showed that affinities of the Aptamers were different,the second structure analysis revealed possible stem-loops for binding to BCR-ABL fusion protein,the affinity of aptamer A2 to BCR-ABL fusion protein was highest with Kd values as low as 72 nmol/L.Conclusion:Aptamers against BCR-ABL fusion protein has been identified by SELEX methods from a 90 bp single stranded DNA library.And provide certain reference for the clinical treatment of chronic myelogenous.