重组腺病毒人内皮抑素的质量标准研究

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2.4×1012 VP/ml,滴度为1.53×1011 IU/ml,比滴度为6.4% IU/VP;成品病毒颗粒数为1.0×1012 VP/ml,滴度为3.75×1010 IU/ml,比滴度为病3.8% IU/VP.以50 MOI重组腺病毒感染HepG2细胞48 h后培养上清人内皮抑素表达量为332 ng/ml.50 MOI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/3×1010 VP.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.

Study of Requirements for Quality Control of Recombinant Adenovirus-Mediated Human Endostatin

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 titer to virus particle for the bulk were 2.4×10 12 VP/ml, 1.53×10 11 IU/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 1.0×10 12 VP/ml, 3.75×10 11 IU/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 332 ng/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 3×10 10 VP recombinant adenovirus products. Other items complied with its corresponding requirements. Conclusion: The methods and requirements had been established for quality control of Adv-human endostatin.