摘要
目的对人ASPP2重组腺病毒制剂进行质量分析,并针对其关键质量特性建立质控方法。方法采用PCR法对重组腺病毒的载体结构特征基因E2B和目的基因ASPP2进行扩增,琼脂糖电泳进行分子量鉴定;采用UV-SDS法测定重组腺病毒的病毒颗粒数;采用组织培养半数感染剂量法(TCID50)测定重组腺病毒的感染滴度;采用Western blot法对重组腺病毒感染靶细胞后目的蛋白ASPP2的表达情况进行检测;通过MTT实验观察重组腺病毒对肝癌细胞的生物学效应;采用A549细胞进行重组腺病毒的复制型病毒检测。结果腺病毒载体E2B基因和目的基因ASPP2的鉴定结果与理论值相符;重组腺病毒的病毒颗粒数为每毫升5.6×10~(12)VP,病毒滴度为每毫升2×10~(11)IU,比活性为3.5%;病毒感染靶细胞24 h后可检测出ASPP2蛋白的表达;重组腺病毒对肝癌细胞具有生长抑制作用;复制型腺病毒(RCA)水平小于1 RCA/3.0×10~(10)VP,符合中国食品药品监督管理局(SFDA)的标准。结论建立了人ASPP2重组腺病毒的关键特征的质量控制方法,为该重组腺病毒质量标准的建立及相关应用奠定了基础。
Aim To analyze the key quality and establish methods for essential quality control of human recombinant ASPP2 adenovirus. Methods The viral structural gene of E2 B and target gene of ASPP2 were identified by PCR; The number of virus particles was measured by UV-SDS methods; Infectious titer was determined by TCID50 assay; Target protein of ASPP2 was observed by Western blot assay; The biological effects of recombinant adenovirus on liver cancer cells were evaluated by MTT as-say; A549 cells were used to check replication of the competent adenovirus( RCA) by the observation of the cytopathic effect.Results PCR analysis of E2 B and ASPP2 was in consistent with theoretical values; Particle numbers of virus were 5. 6 ×10~(12)VP / mL,infectious titer was 2 × 10~(11) IU / mL and specific activity was 3. 5%; ASPP2 protein expression could be detected when cells were infected with virus for 24 h; Growth inhibition of liver cancer cells could be found by adding recombinant ASPP 2adenovirus; The level of RCA was less than 1 RCA /3. 0 × 10~(10) VP,in line with the standards of China Food and Drug Administration( SFDA). Conclusion The quality control methods were established aiming at key characters of human recombinant AS-PP2 adenovirus,which may provide foundations for its quality standard and future applications.
引文
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