乙型肝炎病毒(adr亚型)重组逆转录病毒包装及滴度测定

目的包装乙型肝炎病毒（adr亚型）重组逆转录病毒并进行滴度测定，为下一步构建乙型肝炎病毒（adr亚型）转基因小鼠奠定基础。方法将乙型肝炎病毒（adr）全基因组插入到逆转录病毒载体pLNCL中获得了乙型肝炎（adr）重组逆转录病毒载体，鉴定正反向后，脂质体转染PA317包装细胞，G418抗性压力下进行筛选得到阳性细胞克隆，扩大培养后进行滴度测定，选取产毒率高细胞克隆株，然后扩大培养、浓缩，进行滴度测定。结果分别获得HBV（adr）全基因组正向和反向插入的重组逆转录病毒。正连重组逆转录病毒载体经PA317细胞包装，经筛选、浓缩后滴度为10^6CFU／ml，反连重组逆转录病毒载体经PA317细胞包装，经筛选、浓缩后滴度为10^5CFU／ml。结论PA317细胞包装了含乙型肝炎病毒（adr）全基因组的重组逆转录病毒。

Package of recombinant retrovirus and detection of titre of hepatitis B virus (adr hypotype)

Objective To establish a basement for constructing human hepatitis B virus(adr) transgenic mice model with packaging and determining titer of hepatitis B recombinant retrovirus.Methods The recombinant retroviral vector was obtained by inserted full-length HBV genome(adr)in retroviral vector pLNCL. Following identification of forward and reverse direction connection,the vectors were transfected into PA317 packaging cell line by liposome transfection.Two kinds of cells were screened with G418.G418-resistant PA317 colonies were amplified and concentrated to detect the viral titers.Results Two kinds of hepatitis B recombinant retrovirus were yielded.The titer of one virus with forward direction connection was 10~6 CFU/ml,the titer of another virus was 10~5 CFU/ml. Conclusions The recombinant retroviral with full-length HBV genome(adr) was packed by PA317 cell.