【摘要】 目的建立携骨形态发生蛋白2基因的重组腺相关病毒载体(rAAV-hBMP2-GFP)感染滴度测定的方法,并制备高滴度的rAAV-hBMP2-GFP病毒液,为进一步实验hBMP2诱导大鼠牙槽骨骨缺损修复的基因治疗提供有利的工具。方法通过分析腺相关病毒-HT1080(AAV-HT1080)细胞接种量、培养时间及病毒吸附时间等参数,建立应用荧光计数法测定rAAV-hBMP2-GFP病毒感染滴度的方法,并分析其精密性及检测病毒的稳定性。结果 AAV-HT1080细胞的最佳接种浓度、培养时间及病毒吸附时间分别为3×105个/孔、24h及48h,在此条件下收获的病毒液的感染滴度最高可达3.82×1011/ml,所建立的荧光计数检测方法精密性较好。制备的病毒液的平均感染滴度为3.60×1011/ml,4℃和37℃保存7d及反复冻融5次,稳定性良好。结论建立了rAAV-hBMP2-GFP感染滴度的测定方法,并制备了高滴度及滴度稳定的rAAV-hBMP2-GFP病毒液。更多还原

【Abstract】 Objective To develop a method for determination of infectious titer of the recombinant adeno-associated virus vector with human bone morphonenetic protein 2 and green fluorescent protein gene(rAAV-hBMP2-GFP) and prepare high titer rAAV-hBMP2-GFP for studying the gene therapy of hBMP2 on rat experimental alveolar bone defect. Methods A fluorescence count method for determination of rAAV-hBMP2-GFP titer was developed by optimizing the concentration of AAV-HT1080 cells for inoculation,virus culture time and virus adsorption time;the precision and the stability of the virus detected were analyzed. Results The AAV-HT1080 cell concentration for inoculation,virus culture time and virus adsorption time were 3 × 105 cells /well,24 h and 48 h respectively. In this condition,the infectious titer of rAAV-hBMP2-GFP reached up to 3.82 × 1011 /ml. The developed fluorescence count method showed good precision. The prepared rAAV-hBMP2-GFP was at a mean titer of 3.60 × 1011 /ml,and showed good stability after storage at 4 ℃ and 37 ℃ for 7 d or 5 cycles of freeze-thawing. Conclusion A method for determination of infectious titer of rAAV-hBMP2-GFP is developed,and high titer rAAV-hBMP-2-GFP is prepared.更多还原