重组hPK-5质粒DNA基因治疗制剂的质量标准研究  

Study on quality control methods and requirements of recombinant plasmid DNA expressing human plasminogen kringle 5

作　　者：李永红[1] 张勇朝[1] 袁力勇[1] 史新昌[1] 饶春明[1] 王军志[1] 

机构地区：[1]中国药品生物制品检定所,北京100050

出　　处：《药物分析杂志》2008年第5期661-666,共6页Chinese Journal of Pharmaceutical Analysis

基　　金：国家863高科技研究发展计划项目(2007AA021204)

摘　　要：目的:建立重组 hPK-5质粒 DNA 基因治疗制剂的质量标准和检测方法。方法:采用限制性酶切图谱分析鉴定质粒DNA 结构和 PCR 法鉴定插入的 hPK-5基因作鉴别试验;采用分光光度法测定质粒 DNA 含量和 A_(260)/A_(280)比值;采用琼脂糖凝胶电泳法分析质粒 DNA 的超螺旋构象比例和残留 RNA 含量;采用阴离子交换色谱法分析 HPLC 纯度;采用 ELISA 法测定hPK～5蛋白的表达量;采用内皮细胞迁移法测定 hPK-5蛋白的生物学活性。结果:重组质粒 DNA 的限制性酶切片段大小与理论值一致;插入基因 PCR 扩增片段大小与理论值相符;质粒 DNA 含量为1.12 mg·mL^(-1);A_(260)/A_(280)比值为1.83;琼脂糖凝胶电泳法测定超螺旋构象质粒的比例为95.4%;HPLC 测定超螺旋质粒 DNA 占93.8%,开环质粒 DNA 占6.2%,琼脂糖凝胶电泳法测定木检出 RNA 残留;没有检出其他杂质;以2μg重组 hPK-5质粒转染5×10~5Hela 细胞48 h 后,2 mL 培养上清中 hPK-5蛋白浓度为109 ng·mL^(-1);以培养上清进行内皮细胞迁移试验,转染重组 hPK-5质粒 DNA 组迁移的内皮细胞数明显少于对照绀(P<0.05)。其他项目均符合相应的规定。结论:建立了重组 hPK-5质粒 DNA 基因治疗制剂的检定方法及其质量标准,为有效控制该类产品的质量打下基础。Objective ：To establish quality control methods and requirements for gene therapy products such as recombinant plasmid DNA expressing human plasminogen kringle 5 （hPK -5 ）. Methods：Vector identity was tested by methods such as restriction enzyme mapping and PCR which detected inserted hPK -5 gene on the vector. The content and purity of the plasmid DNA were respectively determined by spectrophotometric absorbence at 260 nm and A260/A280. The supercoiled DNA ratio and RNA residual were analyzed by agarose gel electrophoresis. The purity of plasmid DNA was also determined by anion - exchange HPLC method. The expression level of inserted gene was evaluated by lipo - transfecting Hela cells and detecting concentration of hPK - 5 in the culture supernatant by ELISA method. The potency of expression product was assayed by testing its inhibition effects on the migration of human HMEC endothelial cells. Results：The restriction enzyme fragment length of detected recombinant plasmid DNA conformed to theoretical value. The PCR results of hPK - 5 gene on the vector were consistent with their theoretical expectations. The content of recombinant plasmid DNA was 1.12 mg· mL^-1. The A260/A280 ratio was 1.83. The ratio of supercoiled DNA determined by agarose gel electrophoresis was 95.4%. RNA residual was not detected in the recombinant plasmid DNA analyzed by agarose gel electrophoresis. The results of HPLC purity analysis showed that the ratio of supereoiled plasmid DNA was 93.8% ,the ratio of open - circular plasmid DNA was 6.2% , and other impurity was not detected. After the Hela cells were lipo - transfected by the recombinant plasmid for 48 h,the concentration of hPK-5 in the culture supernatant was 109 ng · mL^-1. When the culture supernatants were analyzed by endothelial cell migration assays, the number of migrated endothelial cells in recombinant hPK - 5 plasmid group was apparently less than the control group （ P 〈 0. 05 ）. Other items complied with their corresponding requirements. Conclusion：The

关 键 词：hPK-5 质粒DNA载体 质量控制 基因治疗 

分 类 号：R917[医药卫生—药物分析学]