A vaccinia replication system for producing recombinant hepatitis C virus.
A vaccinia replication system for producing recombinant hepatitis C virus.
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DOI：
10.3748/wjg.v10.i18.2670
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发表时间：
2004-09
影响因子：
4.3
通讯作者：
Ying-song Wu;Yu-xuan Feng;W. Dong;Yan-ming Zhang;Ming Li
中科院分区：
文献类型：
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作者：
Ying-song Wu;Yu-xuan Feng;W. Dong;Yan-ming Zhang;Ming Li
目的建立以重组痘苗病毒为辅助病毒的丙型肝炎病毒（HCV）细胞培养体系。方法用两种质粒构建重组HCV：一种是将HCV全长cDNA克隆在pOX-T7载体的T7启动子和T7终止子之间，另一种是将HCV多聚蛋白开放阅读框（ORF）直接连接在PSC 59中的牛痘晚期启动子上。将这两种质粒共转染入BHK 21细胞中，然后用vTF 7 -3重组痘苗辅助病毒感染BHK 21细胞。结果培养5d后，荧光定量RT-PCR检测每毫升细胞培养上清中约有3.6 × 10（7）拷贝的HCV RNA。与体外转录HCV基因组RNA或选择性亚基因组HCV RNA分子方法相比，该细胞系统重组HCV的产量提高了100 ~ 1000倍。结论该细胞培养体系能够生产高滴度的重组HCV。
AIM To develop a cell culture system capable of producing high titer hepatitis C virus (HCV) stocks with recombinant vaccinia viruses as helpers. METHODS Two plasmids were used for the generation of recombinant HCV: one containing the full-length HCV cDNA cloned between T7 promoter and T7 terminator of pOCUS-T7 vector, and the other containing the HCV polyprotein open reading frame (ORF) directly linked to a vaccinia late promoter in PSC59. These two plasmids were co-transfected into BHK21 cells, which were then infected with vTF7-3 recombinant vaccinia helper viruses. RESULTS After 5 d of incubation, approximately 3.6X10(7) copies of HCV RNA were present per milliliter of cell culture supernatant, as detected by fluorescence quantitative RT-PCR (FQ-PCR). The yield of recombinant HCV using this cell system increased 100- to 1 000- fold compared to in vitro- transcribed HCV genomic RNA or selective subgenomic HCV RNA molecule method. CONCLUSION This cell culture system is capable of producing high titer recombinant HCV.