指数富集的配体系统进化技术筛选大肠杆菌表达的人CD20胞外蛋白特异性适配体

Screening of the specific aptamer of human CD20 extracellular protein expressed in Escherichia coli by systematic evolution of ligands by exponential enrichment

摘要:

CD20是B细胞淋巴瘤表面标志蛋白，其膜外区是特异性抗体和药物的作用靶点。为了获得靶向CD20的特异性适配体，首先根据密码子简并性对CD20胞外区基因进行优化，使之易于在大肠杆菌中表达；然后将优化后的基因克隆到pGEX-4T-1载体中，转化到大肠杆菌BL21(DE3)中表达；蛋白纯化后用SDS-PAGE和Western blotting进行鉴定；通过指数富集的配体系统进化技术(systematic evolution of ligands by exponential enrichment,SELEX)筛选出与融合蛋白特异性结合的ssDNA适配体，并用流式细胞术检测适配体与CD20的亲和力；再通过细胞毒性试验检测适配体抑制B淋巴瘤细胞的效果。本研究建立了CD20的原核表达方法，获得了特异性结合CD20胞外区蛋白的适配体，为靶向CD20的治疗药物的开发奠定了基础。

Abstract:

CD20 is a surface marker protein of B-cell lymphoma, and its extracellular region is the target of specific antibodies and drugs. To obtain a cheap and easily modified specific preparation targeting CD20, we optimized the gene of CD20 extracellular region according to codon degeneracy to facilitate its expression in Escherichia coli. The optimized gene was cloned into pGEX-4T-1 vector, and the recombinant vector was transformed into E. coli BL21(DE3) for expression. The purified protein was identified by SDS-PAGE and Western blotting. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to screen the ssDNA aptamer that specifically binds to the fusion protein, and the affinity of the aptamer to CD20 was detected by flow cytometry. Then, the cytotoxicity test was carried out to examine the inhibitory effect of the aptamer on B lymphoma cells. In this study, we established the prokaryotic expression method of CD20 and obtained the aptamer specifically binding to the extracellular region of CD20, which laid a foundation for the development of therapeutic drugs targeting CD20.