| 摘要: |
| 目的：建立基因-病毒治疗系统CNHK200-hEndostatin的质量控制方法。方法：分别建立腺病毒蛋白鉴定、腺病毒基因组鉴定、人内皮抑素（hEndostatin）基因鉴定、人内皮抑素表达量检测、腺病毒颗粒数测定、腺病毒滴度和比滴度测定、纯度测定、野生型腺病毒（AdWT）检测、残余宿主DNA含量检测和牛血清蛋白残余量检测等腺病毒质量控制项目的方法，并对纯化的腺病毒样品进行初步检测。结果：建立用SDS-PAGE方法对腺病毒进行蛋白鉴定，用限制性内切酶图谱分析的方法对腺病毒进行基因组鉴定，用PCR方法鉴定人内皮抑素基因，用ELISA方法检测人内皮抑素表达量，用D260法和HPLC方法进行腺病毒颗粒数测定，用D260/D280方法和HPLC方法进行纯度检测，用PCR方法进行野生型腺病毒检测，用杂交方法进行残余宿主DNA含量检测，用反向间接血凝实验测定牛血清蛋白残余量。结果表明建立的方法可有效应用于腺病毒样品检测，并且我们制备的纯化腺病毒样品均符合要求。结论：基本建立了腺病毒CNHK200-hEndostatin的质量控制方法，改良的应用HPLC对腺病毒颗粒数进行定量检测的方法准确迅速，优于传统方法。应用建立的方法对纯化的腺病毒样品进行检测，基本符合临床应用要求。 |
| 关键词: 腺病毒科 内皮抑素类 基因疗法 质量控制 |
| DOI：10.3724/SP.J.1008.2007.01011 |
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| 基金项目:国家自然科学基金国际合作重大项目(30120160823),国家高新技术发展规划(863)重点项目(2001AA217031). |
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| Quality control methods for adenovirus CNHK200-hEndostatin |
| XUE Hui-bin, SHI Jun-xia, ZHU Wen-chuan, CHEN Min, QIAN Qi-jun* |
| (Laboratory of Gene-Viral Therapy，Eastern Hepatobiliary Surgery Hospital，Second Military Medical University, Shanghai 200438, China) |
| Abstract: |
| Objective：To establish a quality control method for Gene-Viral Therapy system CNHK200-hEndostatin. Methods: According to “The Guideline on Quality Control Methods of Human Gene Therapy Products”, a quality control method for adenovirus was set up, which consisted of adenovirus protein identification, genomic identification, hEndostatin gene identification, hEndostatin protein quantitation, adenovirus particle quantitation, virus titer quantitation, determination of particle to PFU ratio, purity detection, AdWT detection, host cell DNA residue detection, bovine serum protein residue detection, etc. The purified adenovirus product of CNHK200-hEndostatin was subjected to the above process. Results: We set up a SDS-PAGE method to identify the adenovirus protein and identified adenovirus genomic DNA by restriction endonuclease enzyme analysis. Human endostatin gene was identified by PCR method and its expression product was quantitated by ELISA. The number of adenovirus particle was quantitated by D260 method and HPLC method. The purity of adenovirus was determined by D260/D280 method and HPLC method. A PCR reaction was introduced to detect AdWT and hybridization method was used in host cell DNA residue detection. Bovine serum protein residue was detected by reverse indirect hemagglutination assay. All these methods were confirmed feasible in adenovirus quality control and our purified adenovirus sample was eligible in all test items.Conclusion: A series of basic methods have been successfully established for quality control of gene-viral therapy system CNHK200-hEndostatin. Our modified method for adenovirus particle quantitation by HPLC is more rapid and accurate than traditional method. The established methods have been approved in testing the purified adenovirus samples. |
| Key words: adenoviridae endostatins gene therapy quality control |
