目的 对无血清悬浮HEK293细胞培养表达狂犬病毒（rabies virus，RV）包膜糖蛋白（glycoprotien，G）的人腺病毒5型（adenovirus type 5，Ad5）重组病毒（Ad5-RVG）的条件进行优化。方法 以Ad5-RVG感染无血清悬浮培养的HEK293细胞，通过检测病毒收获液的感染活性粒子和总病毒粒子，比较不同补加新鲜培养基比例、感染复数、感染细胞密度、病毒培养时间及病毒培养温度等培养条件下的病毒产量，以确定重组病毒的最佳培养条件。结果 Ad5-RVG在无血清悬浮HEK293细胞上的最佳培养条件为：在1.5×10⁶ ml^﹣1的细胞密度下按感染复数10感染Ad5-RVG，并在病毒感染时补加≥50% 293SFM Ⅱ新鲜培养基，于（36±1） ℃、120 r/min培养44~56 h后收获病毒液。结论 确定了Ad5-RVG在无血清悬浮HEK293细胞上的最佳培养条件，为Ad5-RVG的大规模培养提供了重要的研究基础和数据参考。

Objective To optimize the culture conditions of a recombinant adenovirus (Ad) type 5 expressing rabies virus (RV) glycoprotein (G) Ad5-RVG in serum-free suspension HEK293 cells. Methods Serum-free suspension HEK293 cells were infected by Ad5-RVG, and the infectious particle and the total virus particle were detected in the harvest solution under different culture conditions such as the proportion of fresh medium supplemented, multiplicity of infection (MOI), cell density at virus infection, virus culture time and virus culture temperature, etc, to determine the optimal virus culture conditions. Results  The optimal culture conditions of Ad5-RVG in serum-free suspension HEK293 cells were infected by Ad5-RVG at a cell density of 1.5×10⁶ ml^﹣1 with MOI 10, added ≥50% fresh medium of 293SFM Ⅱ along with viral infection, and cultured at （36±1） ℃, 120 r/min for 44-56 h. Conclusion The best culture conditions of Ad5-RVG in serum-free suspension HEK293 cells are determined, which provide an important research basis and data reference for the large-scale culture of Ad5-RVG.