重组PBP2a转肽酶区蛋白核酸适配体的筛选及鉴定

Screening and identification of aptamers against the recombinant transpeptidase domain of PBP2a

目的筛选和鉴定重组青霉素结合蛋白2a(PBP2a)转肽酶区蛋白的核酸适配体。方法利用指数富集的配体系统进化技术(SELEX),以重组PBP2a转肽酶区蛋白为靶标,从单链DNA文库中筛选能与之特异性结合的核酸适配体。筛选产物克隆测序后,对其进行结构分析和特性鉴定。结果经过11轮筛选,单链DNA文库与靶标蛋白的亲和力趋向稳定,将第8、10轮筛选产物克隆测序,对获得的40个适配体进行分析,一级结构分析显示40个适配体并无共同的保守序列,二级结构预测分析表明,其可分为3个家族。其中13号适配体与重组PBP2a蛋白亲和力最高,并能与之特异性结合。结论利用SELEX技术成功筛选出特异性结合重组PBP2a转肽酶区蛋白的核酸适配体,为探索耐甲氧西林金黄色葡萄球菌感染的诊疗新途径奠定基础。

Objective To screen and identify the aptamers of the recombinant transpeptidase domain of PBP2a（penicillin binding protein 2a ,PBP2a） .Methods By using the recombinant transpeptidase domain of PBP2a as the screening target ,oligonucle-otides which were capable of specifically binding to the protein were screened by a random oligonucleotide library through the stem -atic evolution of ligand by exponential enrichment （SELEX ）technique .The ssDNA was cloned and sequenced ,and the secondary structure of aptamer clones was predicted with mfold program .Results After 11 cycles of the selection ,the aptamers which were capable of binding to PBP2a with high affinity have been selected .40 clones from the 8 and 10 cycles were selected randomly and se-quenced .The aptamers obtained had no obvious homology according to their sequences by the sequence alignments ,and the 40 aptamers were classified to three groups according to their secondary structures .The aptamer 13 was found to be specific for the target protein with the highest affinity .Conclusion Aptamers for the recombinant transpeptidase domain of PBP2a with high affili-ty and specificity were successfully screened by SELEX ,which lays a foundation for exploring new ways of diagnosis and treatment of MRSA infection .