重组质粒pUDKH工程菌库的制备及检定

Establishment and Quality Control of Escherichia coli Cell Bank with Recombinant Plasmid pUDKH

目的:pUDKH是由本实验室构建的携带人肝细胞生长因子(HGF)基因的真核表达质粒,具有治疗肢体动脉闭塞病的应用潜能。为了生产制备重组质粒pUDKH,须建立菌种库并对其进行质量检定。方法:将质粒pUDKH转化大肠杆菌DH5α,经筛选后制备成初始菌种库和工作菌种库,对菌种进行革兰染色、电镜、抗生素抗性、遗传稳定性、质粒拷贝数、生化反应等检定。结果与结论:菌种生产pUDKH的产量较高,无支原体及其他微生物污染,40代次内菌种性状、遗传稳定性、质粒拷贝数均稳定,可作为工艺生产用菌种。

Objective： The recombinant plasmid pUDKH, encoding human hepatocyte growth factor（HGF）, is potential for gene therapy of peripheral arterial occlusion disease. Establishment and quality control of E.coli cell bank transformed recombinant plasmid pUDKH are critical steps in plasmid pUDKH manufacture. Methods： The recombinant pUDKH was transformed into E.coli DH5α strain, selected clones were screened for high plasmid production and then amplified to generate a Master Cell Bank. One vial from Master Cell Bank was amplified to produce the Working Cell Bank. Both cell banks were subjected to examination with morphology, antibiotic resistance, genetic stability, copy number and biochemical properties. Results and Conclusion： The bank strains could be used for manufacture of plasmid pUDKH with higher production ability and genetic stability after 40 generational culture, but absence of contaminating bacteria, viruses and other adventitious agent.