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目的应用CS-SELEX(cell surface-system evolution of ligands by exponential enrichment)技术筛选与丙型肝炎病毒(hepatitis C virus,HCV)非结构蛋白4B(non protein 4B,NS4B)特异性结合的ssDNA适配子,建立检测HCV NS4B的酶联寡聚核苷酸吸附试验(enzyme-linked oligonucleotide assay,ELONA)方法。方法构建pDisplay-NS4B质粒,并转染至HepG2细胞中,在G418存在情况下连续筛选1个月,得到单克隆细胞群,经过Western blot以及流式细胞术验证,获得稳定表达NS4B的HepG2细胞。设计并合成随机序列ssDNA文库,以稳定表达NS4B的细胞为正向筛选靶细胞,以HepG2细胞为反筛细胞进行CS-SELEX技术筛选。每轮筛选获得的具有结合NS4B能力的ssDNA适配子,通过PCR扩增得到富集,随后通过不对称PCR获得下一轮筛选的ssDNA文库。经过16轮筛选,将亲和力最高的筛选产物克隆并测序,用ELONA方法检测适配子的亲和力。结果成功构建表面展示表达重组质粒pDisplay-NS4B。分别将该质粒转入HepG2细胞,通过G418筛选获得稳定表达HCV-NS4B的细胞系NS4B-HepG2。对CS-SELEX筛选的各轮适配子库进行亲和力测定,其中第14轮库适配子与NS4B的亲合力最高。分别对第14轮库中的单个适配子进行亲和力测定,以适配子ZN1和ZN5与NS4B的亲合力最高。ZN1和ZN5不仅能结合展示在细胞表面的NS4B蛋白,还能结合丙型肝炎病毒感染细胞(HCV cell culture,HCVcc)中的HCV-NS4B蛋白。结论适配子ZN1和ZN5对NS4B有高亲和力,这对研究HCV-NS4B致癌机制、HCV诊断以及治疗方面有潜在的应用价值。
OBJECTIVE: To screen the ssDNA aptamers that specifically bind to non-protein 4B (NS4B) of hepatitis C virus (HCV) using the cell surface-system evolution of ligands by exponential enrichment (CS-SELEX) , An enzyme-linked oligonucleotide assay (ELONA) was developed to detect HCV NS4B. METHODS: The pDisplay-NS4B plasmid was constructed and transfected into HepG2 cells. The cells were screened continuously for 1 month in the presence of G418 to obtain a monoclonal cell population. The HepG2 cells stably expressing NS4B were obtained by Western blot and flow cytometry. The ssDNA library was designed and synthesized. NS4B cells were stably expressed as target cells, and HepG2 cells were screened by CS-SELEX. The ssDNA aptamers with the ability to bind NS4B were screened each round, enriched by PCR amplification, and the next round of ssDNA libraries was screened by asymmetric PCR. After 16 rounds of screening, the most affinity screening products were cloned and sequenced, and the aptamer affinity was detected by the ELONA method. Results The surface-expressed recombinant plasmid pDisplay-NS4B was successfully constructed. The plasmids were transfected into HepG2 cells respectively, and the NS4B-HepG2 cell line stably expressing HCV-NS4B was obtained by G418 screening. Affinity determination was performed on each round of aptamer library screened by CS-SELEX, with the 14th round of library aptamers having the highest affinity for NS4B. Affinity determination was performed on individual aptamers in the 14th round of library, respectively, and aptamers ZN1 and ZN5 had the highest affinity with NS4B. ZN1 and ZN5 bind to not only the NS4B protein displayed on the cell surface but also the HCV-NS4B protein in HCV cell culture (HCVcc). Conclusion The aptamers ZN1 and ZN5 have high affinity to NS4B, which has potential value in studying the mechanism of HCV-NS4B carcinogenesis, HCV diagnosis and treatment.