重组质粒pVAX1-PENK大规模制备研究  

Large-Scale Production of Recombinant Plasmid pVAX1-PENK

作　　者：蔡珍珍[1,2] 胡春生[2,3] 卢育新[2] 程晓晨[2] 柳伟[2] 张庆林[2] 

机构地区：[1]中南大学药学院,湖南长沙410013 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]北京工业大学生命科学与生物工程学院,北京100124

出　　处：《生物技术通讯》2013年第6期828-832,共5页Letters in Biotechnology

摘　　要：目的：建立质粒pVAX1-PENK的大规模制备2--艺。方法：对大肠杆菌工程菌DH5α-pVAX1-PENK进行补料发酵，利用自行发明的连续碱裂解过程对菌体进行裂解，经超滤浓缩后，用Sepharnse 6 Fast Flow层析柱分离DNA与RNA，再经Plasmidselect Xtra层析柱分离超螺旋质粒DNA与开环或线性质粒DNA，最后经Source 15Q层析柱精制质粒DNA。结果：发酵获得质粒pVAX1-PENK的产率为182mg／L，经碱裂解及层析分离后，最终制备的质粒DNA超螺旋比例大于98％，总回收率为60.5％，纯度（D260nm／D280nm）为1.8～2.0。结论：建立的质粒DNA生产工艺可以制备大量高纯度的质粒DNA，并避免了使用动物源性的酶及有毒试剂。Objective： To explore a large-scale production process of pVAX1-PENK by optimizing fermentation, continuous alkaline lysis and purification procedure. Methods： The fed-batch fermentation process was carried out to produce E.coli DHSα-pVAX1-PENK. The purification process includes continuous alkaline lysis, clarification the lysate, uhrafihration, RNA removing on Sepharose 6 Fast Flow, capturing supercoiled plasmid pVAX1-PENK with Plasmidselect Xtra, and polishing the pVAX1-PENK by Source 15Q. Results： The yield of plasmid pVAX1- PENK from fed-batch fermentation was 182 mg/L. The final production was obtained by alkaline lysis and chromatographic separation. The results show that the supercoiled DNA percentage was 〉98%, the total recovery was 60.5% and the ratio of D260nm/D280nm was between 1.8 and 2.0. Conclusion： The plasmid DNA in large quantities at high purity was produced by the developed procedure without using animal-derived enzyme or toxic solvents.

关 键 词：重组质粒pVAX1-PENK 发酵 连续碱裂解 分离纯化 

分 类 号：Q502[生物学—生物化学] Q78