pUDK-HGF相关药学和基因治疗的实验研究

发布时间：2018-03-23 22:20

本文选题：肝细胞生长因子　切入点：制备　出处：《中国人民解放军军事医学科学院》2006年博士论文

【摘要】：pUDK-HGF是由本实验室构建的携带人肝细胞生长因子(hepatocyte growth factor，HGF)基因真核表达的质粒。实验表明pUDK-HGF可促进肢体缺血部位的血管生成，从而改善肢体功能。pUDK-HGF具有开发成为新的基因药物的前途。pUDK-HGF已获中国专利。“重组质粒-肝细胞生长因子注射液”正在注册申请进行肢体动脉闭塞症的基因治疗临床试验。一、质粒pUDK-HGF的大规模制备和质量分析质粒pUDK-HGF的大规模制备是其药学研究的关键环节，我们建立了pUDK-HGF中试生产流程，主要包括发酵、细菌碱液裂解、Q-Sepharose XL色谱柱富集质粒、Sephacry S1000凝胶色谱分离、Source15Q色谱精制分离、质粒沉淀、重溶、无菌过滤及分装等步骤。并对发酵条件如培养基、pH值、转速、细菌碱性裂解及色谱分离条件进行了优化。所建立的生产流程重复性良好，质粒DNA的回收率较高(约45％)。经质量检定其产品均符合拟定的各项质量检定指标。所有步骤采用一般生物制品制备方法，没有特殊的技术要求和使用特殊的试剂等，存在进一步放大规模的可能性。根据相关国外文献报道及我国基因治疗质量控制指导原则，制定pUDK-HGF质粒制造检定规程，并按此逐项对本实验室中试生产制备的05批次pUDK-HGF的质量进行了分析研究。05批次pUDK-HGF的主要质量如下：pUDK-HGF浓度为2.01mg／ml：A260／A280=1.86；超螺旋pUDK-HGF比例为95.09％；电泳图中未见RNA；残留宿主DNA含量＜0.2％质粒DNA；菌体蛋白质残留含量＜0.1％质粒DNA；限制性酶切图谱与自行制备的对照品一致；PCR扩增到HGF基因片段，大小约2.2kb；未见残余抗生素(卡那霉素)抑菌圈；细菌内毒素＜10EU／mg；pUDK-HGF转染的CHO细胞上清中HGF表达量为39.15(ng／ml)，表达的上清诱导ECV304细胞迁移数高于空质粒对照组2倍。05批次的产品经检验符合拟定的质量标准。该批产品已经中国生物制品检定所复核检定合格。
[Abstract]:PUDK-HGF is a eukaryotic expression plasmid carrying human hepatocyte growth factor-hGFgene constructed in our laboratory. The results show that pUDK-HGF can promote angiogenesis in ischemic region of limbs. Thus, improving limb function. PUDK-HGF has the prospect of being developed as a new gene drug. PUDK-HGF has been patented in China. "Recombinant plasmide-hepatocyte growth factor injection" is applying for clinical trials of gene therapy for extremity arterial occlusive disease. 1. Large-scale preparation and quality analysis of plasmid pUDK-HGF. The preparation of plasmid pUDK-HGF on a large scale is the key link of its pharmacological research. We have established a pilot production process of pUDK-HGF, which mainly includes fermentation, enrichment of plasmid Sephacry S1000 gel chromatographic separation and plasmid precipitation on Q-Sepharose XL chromatographic column. The fermentation conditions such as pH value of culture medium, rotational speed, bacterial alkaline cracking and chromatographic separation conditions were optimized. The recovery rate of plasmid DNA was high (about 45%). The products of plasmid DNA were in accordance with the quality control indexes. All the steps were prepared by general biological products, without special technical requirements and with special reagents, etc. There is the possibility of further scaling up. According to the relevant foreign literature reports and the guiding principles of gene therapy quality control in China, the pUDK-HGF plasmid manufacturing verification regulation was established. According to this, the quality of 05 batches of pUDK-HGF produced in pilot production in our laboratory was analyzed and studied. The main mass of 05 batches of pUDK-HGF was as follows: the concentration of pUDK-HGF was 2.01mg / ml / A260 / A280N 1.86; the ratio of superhelical pUDK-HGF was 95.09; no RNAs were found in electrophoretic map; the content of residual host DNA was < 0.2%. Plasmid DNA; residual protein content of bacteria < 0.1% plasmid DNA; restriction endonuclease digesting map was consistent with the self-prepared reference material to amplify the HGF gene fragment. The size of kanamycin was about 2.2 kb, and no residual antibiotic (kanamycin) inhibition circle was found. The expression of HGF in the supernatant of CHO cells transfected with bacterial endotoxin < 10 EUP / mggpUDK-HGF was 39.15 ng / ml ~ (-1), and the number of ECV304 cell migration induced by the supernatant was 2 times higher than that of the blank plasmid control group. The National Institute for the Control of Biological products is eligible for verification and verification.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346

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