稳定分泌重组HBV载体颗粒细胞系的构建

Stable cell line for secretion of replication-defective hepatitis B virus vector expressing blasticidin resistant gene

目的制备一个持续分泌表达杀稻瘟菌素抗性基因（BsdR）的重组乙型肝炎病毒细胞系。方法删除HBV质粒中各基因的表达，在s基因区插入目的基因BsdR，以构建载体质粒pCH—BsdR；具有G418抗性的无包装信号的HBV辅助质粒pcDNA3．1-CH3142与载体质粒共转染HepG2细胞，经G418和Bsd共筛选，获得细胞克隆，进行系列检测。结果挑选36个细胞克隆，经检测，最终筛选出3个细胞株，具有形成松弛环状DNA的能力，细胞培养上清液中病毒定量分别为4．1×10^6、3．6×10^6、1．2×10^6拷贝／ml。观察到有囊膜的重组HBV颗粒的形成。未检测到野生型HBV的产生。结论该细胞系能够大量制备重组HBV载体颗粒，有助于HBV载体在基因治疗方面的应用和易感细胞系的筛选。

Objective To construct a stable cell line with permanent secretion of recombinant hepatitis B virus（HBV）vector, which express blasticidin resistant gene. Methods Replication-defective HBV vector, pCH- BsdR, which express blasticidin resistance gene was constructed by deleting the HBV genes and inserting the blasticidin resistance gene into the S region. The G418-resistant, the packaging signal deleted HBV helper plasmid, pcDNA3.1-CH3142, and the HBV vector pCH-BsdR were cotransfected into HepG2 cells. Cell clones were selected by the adding of both blasticidin and G418, then serial detection were done.Results After 36 cell clones were picked and expanded. Three cell clones were defined as the best. Quantity of their HBV DNA were 4.1 × 106, 3.6 × 106 and 1.2 × 106 copies/ml, respectively. Enveloped recombinant, but not wild type HBV were confirmed in the culture medium. Conclusions The stable cell lines can realize large preparation of recombinant HBV virions. This will contribute to the use of HBV vector for gene therapy and HBV susceptible cell lines screening.