重组肝靶向干扰素工程菌菌株的遗传稳定性研究  

Genetic stability of recombinant liver-targeting interferon engineered bacteria strains

作　　者：卢雪梅[1,2] 黄演婷[1,2] 汪洁[1,2] 金小宝[1,2] 朱家勇[1,2] 

机构地区：[1]广东药学院药用生物活性物质研究所,广州510006 [2]广东省生物活性药物研究重点实验室,广州510006

出　　处：《生物学杂志》2013年第3期14-16,28,共4页Journal of Biology

基　　金：国家科技部新药创制重大专项资助项目(2013ZX09103003-003)

摘　　要：研究重组肝靶向干扰素工程菌菌株的遗传稳定性,为重组肝靶向干扰素产业化提供依据。在有选择压力(Amp+)条件下,采用平板划线传代法,将重组肝靶向干扰素工程菌菌株连续传代50代,观察菌落和菌体形态,进行生长速度、质粒稳定性、PCR鉴定、限制性内切酶酶切图谱、测序和表达量检测。结果显示:此工程菌在连续传代过程中,保持了大肠杆菌的典型特征,生长速度与原始种子库无明显差异;各代质粒的PCR和酶切图谱正确,传50代后质粒稳定性接近100%,DNA测序未见基因变异。重组肝靶向干扰素表达水平及菌体蛋白的SDS-PAGE图谱均无显著差异。说明肝靶向干扰素工程菌株具有良好的遗传稳定性,为重组肝靶向干扰素的应用奠定了基础。This study aimed to investigate the genetic stability of recombinant plasmid （pET32a-IFN-CSP） in host cell （Escherichia coli BI21 ） for industrialization of recombinant liver-targeting interferon. The engineering strain containing pET32a-IFN-CSP expression vectors was passaged serially under selection pressure （AMP） on LB agar plates for 50 times, and bacterial morphology, growth rate, plasmid stability, PCR identification, restriction enzyme map, sequencing and expressing level were analyzed. The experiment showed that the engineered bacteria maintained the typical characteristics of E. coli in a continuous passage process, no significant difference in the growth rate as the original seed bank; PCR and restriction map of the plasmid were correct, the plasmid stability was nearly 100% and DNA sequencing was no genetic variation after passed through 50 generations. The expression levels of recombinant liver-targeting interferon and the map of SDS-PAGE of cell protein were similar with the origin one. There was no significant difference between the recombinant bacteria of the 50 generations and the original ones, which indicated that recombinant stainhad a high bereditable stability and laid a basis for the application of recombinant liver-targeting interferon.

关 键 词：肝靶向干扰素 重组工程菌 遗传稳定性 

分 类 号：Q78[生物学—分子生物学]