摘要
目的:建立重组腺病毒人内皮抑素的质量标准和检测方法。方法:PCR法鉴定腺病毒载体的E2B区和插入基因,琼脂糖凝胶电泳检查重组病毒DNA酶切图谱。分光光度法测定病毒颗粒数,TCID50法测定病毒感染滴度。感染人肝癌细胞HepG2测定插入基因的表达量,感染人血管内皮细胞HM2测定表达产物的生物学活性。病毒的纯度分析采用A260/A280比值和HPLC法进行。采用A549细胞进行复制型腺病毒的检测。结果:腺病毒载体E2B区和插入基因PCR扩增结果与理论相符,重组病毒DNA的酶切图谱与标准品一致。原液病毒颗粒数为2.4×1012VP/ml,滴度为1.53×1011IU/ml,比滴度为6.4%IU/VP;成品病毒颗粒数为1.0×1012VP/ml,滴度为3.75×1010IU/ml,比滴度为病3.8%IU/VP.以50MOI重组腺病毒感染HepG2细胞48h后培养上清人内皮抑素表达量为332ng/ml。50MOI重组腺病毒对血管内皮细胞生长的抑制率为55%。A260/A280为1.29,HPLC纯度为99.7%。复制型腺病毒为≤1RCA/3×1010VP。其他各项检测指标均符合规定。结论:建立了重组腺病毒人内皮抑素的质量标准及其检测方法,并用于该产品的质量控制。
Objective:To establish the quality control methods and requirements for recombinant adenovirus-human endostatin products. Methods:E2B region on the adenovirus vector and inserted endostatin gene were identified by PCR. The restricted enzyme digestive map of recombinant virus DNA was analysed by agarose gel eletrophoresis. The number of virus particle and infectious titer were determined by UV and TCID50 method respectively. The expression level of inserted gene was analysed by infection of human HepG2 cell. The potency of expression products was determined by its inhibition effects on the proliferation of human HM2 endothelial cell. The purity of Adv-endostatin was analysed by UV and IE-HPLC. The replication competent virus was detected by A549 cell method. Results:The PCR results of E2B region and endostatin gene on the vector were conformed to theoretics. The restricted enzyme digestive map of detected recombinant virus DNA was identical to that of the reference. The number of virus particle, the infectious titer and the ratio of infectious tiler to virus particle for the bulk were 2.4×1012 VP/ml, 1.53×1011IU/ml, and 6.4% IU/VP respectively. The number of virus particle,the infectious titer and the ratio of infectious titer to virus particle for the finished product were 1. 0×1012VP/ml, 3. 75×1011IU/ml and 3. 8% IU/VP respectively. After the HepG2 cell was infected by recombinant virus for 48 hours,the concentration of endostatin in the culture was 332ng/ml. The inhibition rate of 50 MOI recombinant adenovirus to endothelial cell was 55%. The A260nm/A280nm ratio was 1.29. The purity determined by IE-HPLC was 99.7%. There were less than one replication competent virus within 3×1010 VP recombinanl adenovirus producls. Other items complied with its corresponding requirements. Conclusion:The methods and requirements had been established for quality control of Adv-human endostatin.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2005年第2期138-142,共5页
Chinese Journal of Cancer Biotherapy
基金
国家863"十五"计划重大专项(2003 AA223480)
