Vasorin蛋白特异单链DNA适配体的筛选与鉴定

Selection and Identification of ssDNA Aptamers Specific to Vasorin Protein

目的:筛选能特异识别vasorin(VASN)蛋白的单链DNA(ss DNA)适配体并对其进行鉴定。方法:利用SPRSELEX技术筛选VASN蛋白特异ss DNA适配体,通过基因克隆测序、MEME在线软件和RNA structure软件分析富集文库序列并挑选出候选适配体序列,利用EMSA、ELISA、流式细胞术对候选适配体进行特异性与亲和力鉴定。结果:经过5轮SELEX筛选,获得了与VASN蛋白特异结合的ss DNA适配体富集文库,合成候选适配体,经EMSA和ELISA检测分析,证实适配体V4-2能与VASN蛋白特异结合,而不与无关对照牛血清白蛋白结合,其平衡解离常数为281.3±103.7 nmol/L;流式细胞术证实V4-2能够特异识别高表达VASN蛋白的Hep G2细胞。结论:筛选获得特异识别VASN蛋白的ss DNA适配体V4-2。

Objective： To select and identifysingle-stranded DNA（ssDNA） aptamers specific to vasorin（VASN） protein. Methods： SPtl-SELEX technology was used to screen ssDNA aptamers aganist VASN protein. The candidate aptamers were picked out of enriched pools through gene cloning, sequencing and subsequent MEME online software and tlNA structure analysis. The specificity and affinity of the candidates to VASN and VASN-expressing HepG2 cells were further tested by EMSA, ELISA and flow cytometry respectively. Results： The signal of SPtl showed sufficient enrichment after 5 rounds of SELEX selection compared to initial library. EMSA and ELISA re- sults showed that aptamer V4-2 was capable of binding specifically to VASN protein but not to bovine sermn albumin（BSA）, with a dissociation constant of 281.3±103.7 nmol/L. Flow cytometlT results showed that aptamer V4- 2 was capable of binding specifically to HepG2 cells which was highly expressed VSAN protein. Conclusion： We have obtained the ssDNA aptamer specific binding to VASN protein.