抑制肿瘤坏死因子-α的DNA适配子的筛选与鉴定

Screening and Characterization of DNA Aptamers with hTNF-α Binding and Neutralizing Activity

应用SELEX技术筛选能与TNF结合的DNA适配子。化学合成随机寡聚DNA库 ,以TNF为靶蛋白 ,经过12轮SELEX筛选 ,将所得产物克隆、测序。根据所测序列化学合成寡聚DNA适配子 ,用生物素_亲和素_辣根过氧化物酶显色系统检测适配子与TNF的结合活性 ;用鼠L92 9细胞检测适配子拮抗TNF活性。结果显示 ,所筛选到的寡聚DNA能与TNF_α高亲和力结合 ,并能在细胞培养中拮抗TNF_α的细胞毒活性。

Human tumor necrosis factor α (hTNF_α) is one of the most important inflammatory cytokines that acts as a mediator in inflammatory and immune response and plays a key role in host defense against infection. The over expression of hTNF_α is associated with serious consequences, such as shock, hypotension, thrombus, septicemia and even death. It has been implicated in many autoimmune and inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, chronic heart failure and septic shock. Inhibiting the bio_activity of hTNF_α is one of the strategy for the treatment of these diseases. Compared with traditional recombinant protein drugs, small molecule drugs have many advantages, such as high affinity, low immunogenecity and low cost. Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the selection of oligonucleotides that bind with high affinity and specificity to target proteins. Such oligonucleotides are called aptamers, and are potential therapeutics for blocking the activity of pathologically relevant proteins. To obtain oligonucleotide aptamers specifically binding to TNF, a 40nt random DNA combinatorial library flanked by 31nt fixed sequences was chemically synthesized. The random library was amplified with PCR and subjected to selection by SELEX protocol against hTNFα. After incubation of the library with hTNFα, the mixture was blotted onto Immobilon_NC transfer membrane. The no_specific binding was washed away and the hTNFα binding aptamers were eluted and detached from the target protein. The eluted oligo nucleotides were amplified with PCR and served as the DNA library for the next round selection. After 12 rounds of such selection, the selected aptamers were cloned to pGEM_T vector. Positive clones were identified by restriction enzyme digestion and DNA sequencing. Oligo DNA were synthesized according to the sequence data and tested for their activities. Binding activity of the aptamers to hTNFα were detected by ELISA and dot blot with biotin_streptavidin_horseradish peroxidase system. Mouse L929 cells were used to test the anti_hTNFα activity of the DNA aptamers. The aptamers were incubated with hTNFα and added to the L929 cells. The results were read under microscope and with MTT staining. It was shown that these DNA aptamers bound to hTNFα with high affinity, and can inhibit the cytotoxicity of hTNFα on cell culture. The affinity of these aptamers are different and may related to their structure. These ssDNA aptamers are potential for the treatment and diagnosis of hTNFα related diseases.