摘要
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 3.6×10^7 copies of HCV RNA were present per milliliter of cell culture supematant, as detected by fluorescence quantitative RT-PCR(FQ-PCR). The yield of recombinant HCV using this cell system increased 100- to 1000- fold compared to in vitrotranscribed HCV genomic RNA or selective subgenomic HCV RNA molecule method.CONCLUSION: This cell culture system is capable of producing high titer recombinant 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 BHK_(21) cells,which were then infected with vTF7-3 recombinant vaccinia helper viruses. RESULTS:After 5 d of incubation,approximately 3.6×10~7 copies of HCV RNA were present per milliliter of cell culture supematant,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.
基金
Supported by the"863"Program of China,No.2001AA215171
