CAJ | 학술논문

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HCV JFH1及JFH1/GND cDNA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-JFH1和pcDNA3.1-RZ-JFH 1/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1×106 ～3 × 106拷贝/ml的HCV RNA(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HCV JFH1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.
목적 건립가재세포내직접계동HCV복제병차능산생감염성병독과립적HCV전기인조복제자,진행HCV체내외실험연구. 방법 재HCV JFH1급JFH1/GND cDNA서렬량측분별가상핵매서렬여pcDNA3.1재체구건중조질립pcDNA3.1-RZ-JFH1화pcDNA3.1-RZ-JFH 1/GND.용중조질립직접전염Huh7.5세포급진행곤명소서미정맥고압주사.정량실시역전록취합매련반응、세포면역형광염색、면역조직화학염색화혈청학검사분별검측기체내외복제적특성.결과 재16주적검측기내,종전염HCV복제자적Huh7.5세포배양상청액중지속은정지검측출1×106 ～3 × 106고패/ml적HCV RNA(비은정전염),산생적병독과립구유감염성.재미정맥고압주사HCV복제자적곤명소서,검사도단잠적병독혈증,면역조직화학염색미능발현HCV항원양성세포,미검측도혈청중HCV특이성항체.결론 구건적HCV복제자재체외능장기、은정급고효적산생감염성병독과립,단체내실험미능획득예기효과,가능여HCV JFH1병독주적특수성유관.소건립적HCV전기인조복제자가용우HCV병독학급항병독약물사선등방면적연구.
Objective To construct a full-genome hepatitis C virus (HCV) replicon that will allow for direct initiation of replication and generation of infectious viral particles in an in vitro and in vivo cell system.Methods Self-cleaving ribozyme sequences were added to each side of the HCV cDNA clone JFH1 and the replication-deficient clone JFH1/GND,then inserted into the pcDNA3.1 vector downstream of the CMV promoter.The resultant recombinant plasmids,pcDNA3.1-RZ-JFH1 and pcDNA3.1-RZ-JFH1/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 ～ 30 μg/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 × 106 copies/ml) was detected in the supernatant oftransfected Huh7.5 cells up to 16 weeks after transfection.In addition,the viral particles from the supernatant were able to infect na(i)ve Huh7.5 cells.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.