【摘要】 目的探讨8型腺相关病毒的制备方法,初步检测其在体内的表达。方法采用载体质粒、辅助质粒和包装质粒三质粒磷酸钙共沉淀方法转染HEK293细胞,包装病毒,经超声破碎-饱和硫酸铵沉淀-氯化铯梯度离心提取并纯化病毒。肌肉注射感染肌肉组织,检测报告基因增强型绿色荧光蛋白(enhanced green fluoresent prote in,EGFP)在体内的表达活性。结果成功包装制备出8型腺相关病毒,重组病毒能够有效感染肌肉组织,绿色荧光蛋白在肌肉组织中稳定高效表达。结论此实验方法制备病毒的质量能够满足小动物体内实验的要求,为进一步应用其进行基因治疗打下基础。更多还原

【Abstract】 Objective To\ prepare adeno-associated virus serotype 8(AAV8),and examine its expression in vivo.Methods Vector plasmid,helper plasmid and package plasmid were transferred into HEK293 cells by phosphate calcium precipitation.AAV8 was obtained through sonication-saturated ammonium sulfate precipitation-CsCl gradient purification method.After intramuscular injection,AAV8 performance in vivo was examined. Results 2×1012 genome copies/mL of AAV8 was successfully produced,high level of enhanced green fluoresent protein(EGFP)was detected after infecting muscle.Conclusion The quality and quantity of AAV8 prepared by the method can be used to small animal in vivo,which provides a basis for further study in gene therapy.更多还原