双核酶自切割HCV全基因组复制子的构建及其体内外复制的特性  

Construction of a fuH--genome HCV replicon with self-cleaving double ribozyme sequences and characterizationin vitro and in vivo

作　　者：何长龙[1] 刘青山[2] 郭艳[1] 朱研[1] 毛青[1] 兰林[1] 

机构地区：[1]第三军医大学第一附属医院感染病科,重庆400038 [2]第三军医大学第一附属医院康复科,重庆400038

出　　处：《中华肝脏病杂志》2013年第5期348-353,共6页Chinese Journal of Hepatology

基　　金：国家自然科学基金（81071977）l国家教委回国人员启动基金（2010JYB001）,第三军医大学回国人员启动基金（02009XHG08）

摘　　要：目的建立可在细胞内直接启动HCV复制并且能产生感染性病毒颗粒的HCV全基因组复制子，进行HCV体内外实验研究。方法在HCVJFHl及JFHl／GNDcDNA序列两侧分别加上核酶序列与pcDNA3．1载体构建重组质粒pcDNA3．1-RZ-JFHI和pcDNA3．1-RZ-JFHl／GND。用重组质粒直接转染Huh7．5细胞及进行昆明小鼠尾静脉高压注射。定量实时逆转录聚合酶链反应、细胞免疫荧光染色、免疫组织化学染色和血清学检查分别检测其体内外复制的特性。结果在16周的检测期内，从转染HCV复制子的Huh7．5细胞培养上清液中持续稳定地检测出1×10b～3×106拷贝／ml的HCVRNA（非稳定转染），产生的病毒颗粒具有感染性。在尾静脉高压注射HCV复制子的昆明小鼠，检查到短暂的病毒血症，免疫组织化学染色未能发现HCV抗原阳性细胞，未检测到血清中HCV特异性抗体。结论构建的HCV复制子在体外能长期、稳定及高效的产生感染性病毒颗粒，但体内实验未能获得预期效果，可能与HCVJFHl病毒株的特殊性有关。所建立的HCV全基因组复制子可用于HCV病毒学及抗病毒药物筛选等方面的研究。Objective To construct a full-genome hepatitis C virus （HCV） replicon that will allow for direct initiation of replication and generation of infections viral particles in an in vitro and in vivo cell system. Methods Self-cleaving ribozyme sequences were added to each side of the HCV eDNA clone JFH1 and the replication-deficient done JFH1/GND, then inserted into the pcDNA3.1 vector downstream of the CMV promoter. The resultant recombinant plasmids, PeDNA3.1-RZ-JFH 1 and pcDNA3.1-RZ-JFH 1/GND, were tested for activity in vitro and in vivo by transiently transfecting into Huh7.5 cells （5 μg/100 mm culture dish） and injecting by high-pressure tail vein injection into Kunming mice （10 - 30μg/mouse）. Quantitative reverse transcription-PCR, immunofluorescence, immunohistochemistry, and serological testing were performed to determine the replication ability and assess the properties of the recombinant plasmids in the two systems. Results HCV RNA （1 - 3 x 106 copies/ml） was detected in the supematant of transfected Huh7.5 cells up to 16 weeks after transfection. In addition, the viral particles from the supematant were able to infect naive Huh7.5 ceils. However, only transient viremia was achieved upon tail vein injection of the plasmid, and no HCV antigen-positive cells were detected by immunohistochemistry nor HCV-specific antibodies by serological testing. Conclusion The constructed HCV replicon was capable of stable expression in cultured cells and of efficiently generating infectious viral particles in the in vitro system over a long period. However,the HCV replicon did not show infective characteristics in an in vivo mouse system. The full-length HCV replicon may represent a useful tool for in vitro study of HCV pathological mechanisms, possibly including anti-HCV drug screening.

关 键 词：肝炎病毒 丙型 全基因组复制子 HUH7 5细胞 昆明小鼠 

分 类 号：R512.63[医药卫生—内科学]