中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2011年
6期
523-527
,共5页
高博%贾帅争%彭剑淳%王怡%樊炜%李银太%詹林盛%许金波
高博%賈帥爭%彭劍淳%王怡%樊煒%李銀太%詹林盛%許金波
고박%가수쟁%팽검순%왕이%번위%리은태%첨림성%허금파
丙型肝炎病毒%萤火虫荧光素酶%细胞模型%复制
丙型肝炎病毒%螢火蟲熒光素酶%細胞模型%複製
병형간염병독%형화충형광소매%세포모형%복제
HCV%Firefly luciferase%Cell model%Replication
目的 建立稳定的报告基因标记的HCV全基因组复制细胞模型,对细胞内病毒蛋白和核酸的表达水平进行定量.方法 把neo抗性基因插入到Luc-JC1中,构建Luc-JC.将体外转录的Luc-JC RNA转染Huh7细胞,经筛选后获得的G418抗性克隆,通过荧光素酶活性检测、Western blot和HCV NS3/4A对eYFP-MAVS的切割试验进行鉴定.用IFN-α处理筛选到的细胞,观察细胞中荧光素酶活性、HCV相关蛋白和RNA的变化,对复制细胞在抗病毒药物评价方面的应用进行初步验证.结果 G418加压筛选得到了多个含HCV全基因组的细胞克隆.在细胞克隆中,荧光素酶活性检测观察到萤火虫荧光素酶;Western blot检测到HCV NS3和NS5A蛋白的表达;由于NS3/4A的切割,eYFP-MAVS的定位由线粒体转移到细胞质.IFN-α处理阳性克隆细胞,荧光素酶活性、HCV蛋白表达和RNA水平明显降低.结论成功建立稳定的报告基因标记的HCV全基因组复制细胞模型,报告基因能用来指示细胞内HCV蛋白和RNA的表达水平,为进一步开展HCV致病机制研究和治疗药物筛选奠定了基础.
目的 建立穩定的報告基因標記的HCV全基因組複製細胞模型,對細胞內病毒蛋白和覈痠的錶達水平進行定量.方法 把neo抗性基因插入到Luc-JC1中,構建Luc-JC.將體外轉錄的Luc-JC RNA轉染Huh7細胞,經篩選後穫得的G418抗性剋隆,通過熒光素酶活性檢測、Western blot和HCV NS3/4A對eYFP-MAVS的切割試驗進行鑒定.用IFN-α處理篩選到的細胞,觀察細胞中熒光素酶活性、HCV相關蛋白和RNA的變化,對複製細胞在抗病毒藥物評價方麵的應用進行初步驗證.結果 G418加壓篩選得到瞭多箇含HCV全基因組的細胞剋隆.在細胞剋隆中,熒光素酶活性檢測觀察到螢火蟲熒光素酶;Western blot檢測到HCV NS3和NS5A蛋白的錶達;由于NS3/4A的切割,eYFP-MAVS的定位由線粒體轉移到細胞質.IFN-α處理暘性剋隆細胞,熒光素酶活性、HCV蛋白錶達和RNA水平明顯降低.結論成功建立穩定的報告基因標記的HCV全基因組複製細胞模型,報告基因能用來指示細胞內HCV蛋白和RNA的錶達水平,為進一步開展HCV緻病機製研究和治療藥物篩選奠定瞭基礎.
목적 건립은정적보고기인표기적HCV전기인조복제세포모형,대세포내병독단백화핵산적표체수평진행정량.방법 파neo항성기인삽입도Luc-JC1중,구건Luc-JC.장체외전록적Luc-JC RNA전염Huh7세포,경사선후획득적G418항성극륭,통과형광소매활성검측、Western blot화HCV NS3/4A대eYFP-MAVS적절할시험진행감정.용IFN-α처리사선도적세포,관찰세포중형광소매활성、HCV상관단백화RNA적변화,대복제세포재항병독약물평개방면적응용진행초보험증.결과 G418가압사선득도료다개함HCV전기인조적세포극륭.재세포극륭중,형광소매활성검측관찰도형화충형광소매;Western blot검측도HCV NS3화NS5A단백적표체;유우NS3/4A적절할,eYFP-MAVS적정위유선립체전이도세포질.IFN-α처리양성극륭세포,형광소매활성、HCV단백표체화RNA수평명현강저.결론성공건립은정적보고기인표기적HCV전기인조복제세포모형,보고기인능용래지시세포내HCV단백화RNA적표체수평,위진일보개전HCV치병궤제연구화치료약물사선전정료기출.
Objective To establish a stable HCV full-length genome replication cell model which is labeled with reporter gene and easyly to quantify intracellular HCV proteins and RNA level. Methodsneo gene was inserted into Luc-JC1 to make Luc-JC construct. Luc-JC RNA was obtained by in vitro transcription and then delivered into Huh7 cells by transfection. G418-resistant clones of Huh7 cells were obtained by selection. Clones of HCV full-length genome replication cell were confirmed by luciferase activity assay, Western blot and cleaveage of eYFP-MAVS by HCV NS3/4A protease. Then, HCV replication cell colonies were treated by different dose IFN-α in order to observe the change of luciferase activity, HCV protein and RNA level. Results At 3-4 weeks post-transfection, visible colonies were selected and stained by crystal violet. Luciferase activity and HCV NS3, NS5A protein were detected by luciferase activity assay and Western blot, respectively. Subcellular localization of eYFP-MAVS transferred from mitochondria to cytoplasms by cleavage of NS3/4A protease in cell colonies. Luciferase activity, HCV protein and RNA diminished obviously after IFN-α treatment. Conclusion A stable HCV full-length genome replication cell model labeled by reporter gene was successfully established and reporter activity can be used to indicate level of HCV proteins and RNA in cells. This cell model is a useful tool for the study on HCV pathogenesis and the screening of antiviral drugs.