【目的】探究关键病毒基因和辅助基因对rAAV生产的影响以指导病毒基因表达框的理性设计和工艺优化,实现rAAV生产效率的提升。【方法】首先,明确用于生产rAAV的关键基因元件rep和cap基因的组成和比例,以及辅助病毒基因的关键序列对rAAV...【目的】探究关键病毒基因和辅助基因对rAAV生产的影响以指导病毒基因表达框的理性设计和工艺优化,实现rAAV生产效率的提升。【方法】首先,明确用于生产rAAV的关键基因元件rep和cap基因的组成和比例,以及辅助病毒基因的关键序列对rAAV生产的影响。然后,据此理性设计质粒上rAAV病毒基因表达框,构建新型三质粒系统。最后,通过对新型三质粒系统关键工艺参数进行优化,建立基于新型三质粒系统的rAAV高效生产工艺。【结果】合理的rep基因和cap基因表达比例对rAAV生产至关重要,辅助病毒基因表达框中仅保留关键蛋白序列E4orf6(adenovirus early region 4 open reading frame 6,E4orf6)和DBP(DNA binding protein,DBP)即可实现同样的rAAV生产效率。据此结果重新设计的新型三质粒系统瞬时转染人胚胎肾细胞(human embryonic kidney 293 cells,HEK293)时,rAAV基因组滴度较传统三质粒系统提高2.6倍。在进一步优化该系统的转染参数后,基因组滴度达到7.8×10^(11)vg/mL,单细胞产毒量达到1.5×10^(5)vg/cell,相较于优化前分别提高3.7倍和2.4倍。【结论】通过探究rAAV病毒基因和辅助病毒基因的组成与比例对病毒载体包装的影响,重新设计了rAAV的瞬时表达系统,优化并建立了新型三质粒系统的rAAV生产工艺,显著提高了rAAV的产量,其中基因组滴度提高16倍,单细胞产量提高10倍,为rAAV的高效工业化生产奠定了基础。展开更多
We constructed an expression cassette of the organophosphorus pesticide degrading （opal） gene under the control of the E8 promoter. Then opd was transformed into tomato fruit using an agroinfiltration transient expr...We constructed an expression cassette of the organophosphorus pesticide degrading （opal） gene under the control of the E8 promoter. Then opd was transformed into tomato fruit using an agroinfiltration transient expression system. β-Glucuronidase （GUS） staining, reverse transcription-polymerase chain reaction （RT-PCR）, wavelength scanning, and fluorescent reaction were performed to examine the expression of the opd gene and the hydrolysis activity on coumaphos of organo- phosphorus hydrolase （OPH） in tomato fruit. The results show that the agroinfiltrated tomato fruit-expressed OPH had the maximum hydrolysis activity of about 11.59 Uhng total soluble protein. These results will allow us to focus on breeding transgenic plants that could not only enhance the degrading capability of fruit and but also hold no negative effects on pest control when spraying organophosphorus pesticides onto the seedlings in fields.展开更多
文摘【目的】探究关键病毒基因和辅助基因对rAAV生产的影响以指导病毒基因表达框的理性设计和工艺优化,实现rAAV生产效率的提升。【方法】首先,明确用于生产rAAV的关键基因元件rep和cap基因的组成和比例,以及辅助病毒基因的关键序列对rAAV生产的影响。然后,据此理性设计质粒上rAAV病毒基因表达框,构建新型三质粒系统。最后,通过对新型三质粒系统关键工艺参数进行优化,建立基于新型三质粒系统的rAAV高效生产工艺。【结果】合理的rep基因和cap基因表达比例对rAAV生产至关重要,辅助病毒基因表达框中仅保留关键蛋白序列E4orf6(adenovirus early region 4 open reading frame 6,E4orf6)和DBP(DNA binding protein,DBP)即可实现同样的rAAV生产效率。据此结果重新设计的新型三质粒系统瞬时转染人胚胎肾细胞(human embryonic kidney 293 cells,HEK293)时,rAAV基因组滴度较传统三质粒系统提高2.6倍。在进一步优化该系统的转染参数后,基因组滴度达到7.8×10^(11)vg/mL,单细胞产毒量达到1.5×10^(5)vg/cell,相较于优化前分别提高3.7倍和2.4倍。【结论】通过探究rAAV病毒基因和辅助病毒基因的组成与比例对病毒载体包装的影响,重新设计了rAAV的瞬时表达系统,优化并建立了新型三质粒系统的rAAV生产工艺,显著提高了rAAV的产量,其中基因组滴度提高16倍,单细胞产量提高10倍,为rAAV的高效工业化生产奠定了基础。
基金Project supported by the National Key Technology R&D Program of China(No.2007BAD59B06)the International Science and Technology Cooperation Program of China(No.2007DFA31260)
文摘We constructed an expression cassette of the organophosphorus pesticide degrading （opal） gene under the control of the E8 promoter. Then opd was transformed into tomato fruit using an agroinfiltration transient expression system. β-Glucuronidase （GUS） staining, reverse transcription-polymerase chain reaction （RT-PCR）, wavelength scanning, and fluorescent reaction were performed to examine the expression of the opd gene and the hydrolysis activity on coumaphos of organo- phosphorus hydrolase （OPH） in tomato fruit. The results show that the agroinfiltrated tomato fruit-expressed OPH had the maximum hydrolysis activity of about 11.59 Uhng total soluble protein. These results will allow us to focus on breeding transgenic plants that could not only enhance the degrading capability of fruit and but also hold no negative effects on pest control when spraying organophosphorus pesticides onto the seedlings in fields.
