小鼠神经生长因子基因治疗型DNA质粒的质量控制

Quality Control of Mouse Nerve Growth Factor DNA Plasmid for Gene Therapy

目的对小鼠神经生长因子(NGF)基因治疗型DNA质粒进行质量控制。方法用酶切鉴定法和PCR法进行DNA质粒的结构确认,鸡胚背根神经节法和免疫印迹测定DNA质粒表达产物的生物学活性,分光光度法测定浓度,琼脂糖凝胶电泳法和DNA-NPR-HPLC法测定纯度,气相色谱法测定乙醇和异丙醇残留量,琼脂糖凝胶电泳法测定RNA残留量,其余检测项目按《中国药典》三部(2005版)规定进行。结果用上述方法对原液和成品进行了检定,各项指标均符合《预防用DNA疫苗临床前研究技术指导原则》和《中国药典》三部(2005版)的要求。结论所采用的质控方法和质量标准能够保证该DNA质粒的安全、有效,可用于治疗型DNA质粒的质控。

Objective To control the quality of mouse nerve growth factor （ NGF ） DNA plasmid for gene therapy. Methods Confirm the structure of mouse NGF DNA plasmid by restriction analysis and PCR. The expressed product of NGF DNA plasmid was determined for biological activity by chick embryonic dorsal root ganglion Axon assay and dot blot, for concentration by spectrophotometry, for purity by agarose gel electrophoresis and DNA-NPR-HPLC, for residual ethanol and isopropanol contents by gas chromatography, and for residual RNA by agarose gel electrophoresis. The other control tests were performed according to the requirements in Chinese Pharmacopoiea（ 2005 edition ）. Results All the results of control tests met the requirements in Chinese Pharnuwopoiea （2005 edition） and in Guidelines for Preclinical Study of Prophylactic DNA Vaccine issued by SFDA. Conclusion The methods and standards used in this paper ensured the safety and effectiveness of mouse NGF DNA plasmid, and might be used for the quality control of therapeutic DNA plasmid.