重组腺相关病毒2型/人凝血因子IX的质量研究重组腺相关病毒2型/人凝血因子IX的质量研究

高凯; 王军志; 饶春明; 吴小兵

重组腺相关病毒2型/人凝血因子IX的质量研究重组腺相关病毒2型/人凝血因子IX的质量研究

Quality control of recombinant adeno-associated virus type 2/human blood coagulation factor IX

摘要

摘要: 目的研究并建立重组腺相关病毒2型/人凝血因子IX(recombinant adeno-associated virus type 2/human blood coagulation factor IX,rAAV-2/hFIX)的质量标准。方法采用PCR法确认病毒所携带的重组核酸结构和测定辅助病毒(helper virus)和野生型腺相关病毒（wtAAV）的残留片段。SDS-PAGE电泳测定病毒外壳蛋白分子量及纯度，TSK gel SP-NPR阳离子交换柱系统测定病毒颗粒纯度。以斑点杂交法测定病毒颗粒数。一期法于IX因子基因剔除小鼠体内测定rAAV-2/hFIX生物学活性，并通过ELISA法测定感染BHK-21细胞后hFIX的表达量。结果PCR法确证病毒的重组核酸结构与构建预期相同；在1×10⁷ VG的rAAV-2/hFIX颗粒中，残留辅助病毒的基因片段数少于1个拷贝；在1×10⁸ VG的rAAV-2/hFIX颗粒中，野生型AAV-2基因片段数少于1个拷贝。病毒颗粒及外壳蛋白纯度均大于98%，病毒颗粒数大于1.0×10₁₅VG·L^-1(virus genome·L^-1)。IX因子剔除小鼠肌肉注射病毒后21 d，小鼠血液中人凝血因子IX活性达到大于正常人因子IX活性的15%，IX因子的体外表达水平大于20.0 μg·L^-1。其他各项检测指标均符合规定。结论建立了rAAV-2/hFIX的质量标准，用于控制产品质量。

Abstract: AimTo establish quality control requirements and methods for recombinant adeno-associated virus（rAAV）type 2/human blood coagulation factor IX (rAAV-2/hFIX). MethodsIdentification of rAAV genome fragments, potential contaminants including wild type AAV(wtAAV) and helper virus, were detected by PCR. Purity of rAAV-2/hFIX was analyzed by cation-exchange HPLC and SDS-PAGE. Virus partical numbers were performed by dot blot assay. hFIX expression was demonstrated by ELISA and potency of hFIX was verified by APTT. ResultsIdentity of rAAV-2/hFIX was proved. Residues of wtAAV and helper virus were conformed to requirements. Purity of rAAV-2/hFIX were more than 98%. Partical numbers of rAAV-2/hFIX were more than 1.0×10₁₅VG·L^-1. hFIX expression was more than 20.0 μg·L^-1. hFIX potency was verified by APTT following rAAV-2/hFIX injected to FIX gene knockout mice, potency results conformed to requirements. ConclusionThe methods and requirements had been established for quality control of rAAV-2/hFIX.

HTML全文

参考文献(0)

施引文献

资源附件(0)