人ASPP2重组腺病毒质量控制研究
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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.
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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[10]中国生物制品标准化委员会.人基因治疗研究和制剂质量控制技术指导原则[R].北京:国家食品药品监督管理局,2003.[10]The State Biological Products Standardization Commission of the People’s Republic of China.Guidance for human gene therapy research and its products quality control[R].Beijing:SFDA,2003.
[11]Chen G X,Zhang S,He X H,et al.Clinical utility of recombinant adenoviral human p53 gene therapy:current perspectives[J].Onco Targets Ther,2014,7:1901-9.
[12]Trigiante G,Lu X.ASPP[corrected]and cancer[J].Nat Rev Cancer,2006,6(3):217-26.
[13]Sullivan A,Lu X,ASPP:a new family of oncogenes and tumour suppressor genes[J].Br J Cancer,2007,96(2):196-200.
[14]林建伟,王饶高.第二届药品技术审评研讨会论文集[C].北京,2003.[14]Lin J W,Wang R G.Proceedings of the second symposium on drug technical review[C].Beijing,2003.
[2]席聪,安睿,李海勋,等.r AAV-PR39-ADM防治大鼠脑缺血/再灌注损伤的研究[J].中国药理学通报,2015,31(5):641-7.[2]Xi C,An R,Li H X,et al.Research on prevention and treatment effect of Raav-PR39-ADM in cerebral ischemia reperfusion injury in rats[J].Chin Pharmacol Bull,2015,31(5):641-7.
[3]Samuels-Lev Y,O'Connor D J,Bergamaschi D,et al.ASPP proteins specifically stimulate the apoptotic function of p53[J].Mol Cell,2001,8(4):781-94.
[4]Gorina S,Pavletich N P.Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2[J].Science,1996,274(5289):1001-5.
[5]Vives V,Su J,Zhong S.ASPP2 is a haploinsufficient tumor suppressor that cooperates with p53 to suppress tumor growth[J].Genes Dev,2006,20(10):1262-7.
[6]Kampa K M,Acoba J D,Chen D,et al.Apoptosis-stimulating protein of p53(ASPP2)heterozygous mice are tumor-prone and have attenuated cellular damage-response thresholds[J].Proc Natl Acad Sci USA,2009,106(11):4390-5.
[7]Ma L,Chen Z M,Li X Y,et al.Nucleostemin and ASPP2 expression is correlated with pituitary adenoma proliferation[J].Oncol Lett,2013,6(5):1313-8.
[8]Mak V C,Lee L,Siu M K,et al.Downregulation of ASPP2 in choriocarcinoma contributes to increased migratory potential through Src signaling pathway activation[J].Carcinogenesis,2013,34(9):2170-7.
[9]Schittenhelm M M,Illing B,Ahmut F,et al.Attenuated expression of apoptosis stimulating protein of p53-2(ASPP2)in human acute leukemia is associated with therapy failure[J].PLo S One,2013,8(11):e80193.
[10]中国生物制品标准化委员会.人基因治疗研究和制剂质量控制技术指导原则[R].北京:国家食品药品监督管理局,2003.[10]The State Biological Products Standardization Commission of the People’s Republic of China.Guidance for human gene therapy research and its products quality control[R].Beijing:SFDA,2003.
[11]Chen G X,Zhang S,He X H,et al.Clinical utility of recombinant adenoviral human p53 gene therapy:current perspectives[J].Onco Targets Ther,2014,7:1901-9.
[12]Trigiante G,Lu X.ASPP[corrected]and cancer[J].Nat Rev Cancer,2006,6(3):217-26.
[13]Sullivan A,Lu X,ASPP:a new family of oncogenes and tumour suppressor genes[J].Br J Cancer,2007,96(2):196-200.
[14]林建伟,王饶高.第二届药品技术审评研讨会论文集[C].北京,2003.[14]Lin J W,Wang R G.Proceedings of the second symposium on drug technical review[C].Beijing,2003.