丙型肝炎病毒单链RNA免疫活性的研究  

Study on immunological activity of single strand RNA of hepatitis C virus

作　　者：程娜[1] 章意亮[1] 魏伟[1] 王凯慧[1] 孙树汉[1] 

机构地区：[1]第二军医大学基础部医学遗传学教研室,上海200433

出　　处：《第二军医大学学报》2010年第9期929-932,共4页Academic Journal of Second Military Medical University

基　　金：国家高技术研究发展计划("863"计划;2006AA02A238);全军"十一五"科技攻关项目(06G067)~~

摘　　要：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 the effective immunostimulatory sequences from hepatitis C virus（HCV） single strand RNA（ssRNA）, and to identify the proteins that can bind to the sequences specifically. Methods Four oligonucleotide sequences （HCV-ORNs） derived from HCV ssRNA were synthesized, and the cytokines induced by them were detected using enzyme linked immunosorbent assay （ELISA）, so as to identify the effective immunostimulatory sequences. Then proteins binding to the sequence（s） specifically were identified using improved electrophoresis mobility shift assay （EMSA） combined with electrospray ionization mass spectrum （ESI-MS）. Results One of the HCV-ORNs, ORNHCVtail, induced higher levels of TNF-α and IFN-α （P〈0.01）. Totally 83 groups of proteins were identified that can bind to ORNHCVtail; the proteins included TLR3（Toll-like receptor 3）, NLRC3（NLR family, CARD domain-containing 3）, and NLRC4（NLR family, CARD domain-containing 4）. Conclusion We have successfully screened out effective immunostimulatory sequence of HCV ssRNA and the proteins that can bind to the sequence specifically. Our findings in the present study lay a foundation for mechanism research of immunity stimulated by HCV infection.

关 键 词：丙型肝炎病毒 单链RNA 免疫激活序列 RNA结合蛋白 

分 类 号：R373.21[医药卫生—病原生物学]