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目的筛选丙型肝炎病毒(HCV)单链RNA(single strand RNA,ssRNA)序列中有效的免疫激活序列,并进一步确定与免疫激活序列特异结合的蛋白。方法筛选并合成4条HCV ssRNA序列中的寡聚核苷酸序列(HCV-ORNs),通过ELISA法检测其诱导的细胞因子表达情况,确定有效免疫激活序列。然后通过改良型凝胶阻滞实验及质谱分析技术筛选与该序列特异结合的蛋白。结果在4个HCV-ORNs中,ORNHCVtail可有效诱导细胞因子TNF-α、IFN-α升高(P<0.01)。与ORNHCVtail特异结合的蛋白条带经质谱鉴定共鉴定出83种蛋白,其中可能与HCV ssRNA激活免疫反应相关的蛋白包括:TLR3(Toll-like receptor3),NLRC3(NLR family,CARD domain-containing3),NLRC4(NLR family,CARD domain-containing4,又称IPAF)。结论成功筛选出HCV ssRNA序列中有效的免疫激活序列,并筛选出与其特异结合的蛋白,为HCV激活天然免疫反应的机制研究奠定了基础。
OBJECTIVE: To screen for effective immune activation sequences of single strand RNA (ssRNA) sequences of hepatitis C virus (HCV) and to further identify proteins that specifically bind to immune activation sequences. Methods The four HCV ssRNA sequences (HCV-ORNs) were screened and synthesized. The expression of cytokines was detected by ELISA and the effective immune activation sequence was determined. Then, the protein that binds specifically with this sequence was screened by modified gel retardation assay and mass spectrometry. Results Of the four HCV-ORNs, ORNHCVtail effectively induced the increase of cytokines TNF-α and IFN-α (P <0.01). A total of 83 proteins were identified by mass spectrometry. The proteins that may be involved in HCV ssRNA activation of immune response include: TLR3 (Toll-like receptor3), NLRC3 (NLR family, CARD domain-containing3) NLRC4 (NLR family, CARD domain-containing4, also known as IPAF). Conclusion The successful screening of HCV ssRNA sequences and screening out the specific binding proteins have laid the foundation for the study on the mechanism of HCV innate immune response.