摘要
肿瘤治疗性DNA疫苗pSFVK1-NMM由于采用了基于塞姆利基森林病毒(semliki for estvirus,SFV)的复制子系统,使得其大小超过15 kb,所以需要对该疫苗生产用菌株进行筛选后才能建立三级种子库。本研究首先对不同构型的pSFVK1-NMM质粒进行了电泳分析,对该质粒的超螺旋构型、线性构型、开环构型和碱变性构型的电泳特征进行了描述。然后分别在37,34,30℃培养温度下对比该质粒结构稳定性的差异,发现30℃能提供该质粒稳定的复制环境,为工程菌合适的培养温度,同时也对因高温培养所产生的截短型DNA进行了电泳分析。最后通过30℃条件下的传代培养对21株含有质粒的重组大肠杆菌进行筛选,比较第5,10,15,20代的质粒电泳结果,获得1株能够稳定培养20代的菌株作为生产用工程菌,后续可用于三级种子库的建立。
The tumor therapeutic DNA vaccine pSFVK1-NMM is over 15 kb in size due to its replication system based on semliki forest virus(SFV).It is necessary to screen the strains for vaccine production before establishing a three-tier seed bank.In this study,the pSFVK1-NMM plasmids with different configurations were analysised by electrophoresis,and the electrophoretic characteristics of the supercoiled,linear,open circular and alkali-denatured configurations of the plasmid were described.Then the differences in the structural stability of the plasmid were compared at 37,34,30℃respectively.It was found that 30℃could provide a stable replication environment for the plasmid,which was a suitable culture temperature for engineering strain,and the truncated DNA caused by high temperature was also analyzed by electrophoresis.Finally,21 strains of recombinant Escherichia coli containing plasmids were screened by subculture at 30℃.Electrophoresis results of the 5th,10th,15th and 20th generations were compared,and one strain that could be stably cultured for 20 generations was obtained as the engineering strain for production,which could be used for the establishment of a three-tier seed bank.
作者
柯尊阳
孔垂占
于继云
王宇
KE Zunyang;KONG Chuizhan;YU Jiyun;WANG Yu(School of Pharmaceutical Sciences,Wuhan University,Wuhan 430061,China;Gu’an Dingtai Haigui Biotechnology Co.,Ltd.,Langfang,Heibei 065500,China)
出处
《中国科技论文在线精品论文》
2022年第3期311-317,共7页
Highlights of Sciencepaper Online
基金
河北省科技厅重点研发计划(20372404D)
关键词
生物工程
DNA疫苗
菌株筛选
连续传代
bioengineering
DNA vaccine
strain screening
continuous cultivation
