高密度培养工艺对质粒菌质粒产量的影响  

High Density Culture Technology of Plasmid Bacteria

作　　者：张乐 王润杨 石晓太 胡翰 汪洋 刘滨磊 ZHANG Le;WANG Runyang;SHI Xiaotai;HU Han;WANG Yang;LIU Binlei(School of Biological Engineering and Food,Hubei Univ.of Tech.,Wuhan 430068,China)

机构地区：[1]湖北工业大学生物工程与食品学院,湖北武汉430068

出　　处：《湖北工业大学学报》2025年第1期70-75,共6页Journal of Hubei University of Technology

摘　　要：为通过优化质粒pUC57的复制起始位点,提高其在宿主菌中的拷贝数,利用随机突变PCR技术对质粒pUC57的复制起始位点进行了定向突变,发现突变后的质粒pUC57-Mu-Ori-kana(Mu代表突变)显示更高的拷贝数。采用分子克隆技术中的同源重组方法,将pUC57-Mu-Ori-kana作为载体,连接新的目的基因片段。通过使用商品化感受态细胞DH10B对质粒进行转化,发现质粒产量显著提升。经过传代稳定性验证,进一步在生物反应器中使用改良的MLB培养基对新质粒菌进行放大培养。最终,从发酵罐中取得的样本显示,质粒的终产量可达到600 mg/L。突变后的复制起始位点显著提升了质粒的拷贝数,挑取阳性单克隆测序,最终获得了两个不同的质粒目的片段序列。通过同源重组技术,将突变后的片段和未突变的片段与原始载体连接起来,对比发现,在相同的转化情况和提取工艺条件下,pUC57-Mu-Ori-kana提取量是原始pUC57-kana的1.5倍。此外,DH10B-pUC57-Mu-Ori-kana在生物反应器放大培养后,也得到了令人满意的产量。This study first mutated the replication origin of plasmid pUC57 using a random mutation PCR kit,and found that the plasmid copy number of the mutated pUC57 Mu Ori kana(Mu:Mutation)increased.Using molecular cloning technique homologous recombination,plasmid pUC57 Mu Ori kana was used as a vector to connect new target fragments.The plasmid was transformed using commercialized competent cells DH10B and found that the plasmid yield increased.After the stability verification of the new plasmid bacteria through passage,the bioreactor and modified MLB medium were used for amplification culture.Finally,the bacterial liquid in the fermenter was sampled,and the final yield of plasmid in the tank reached 600 mg/L.By mutating the replication origin,the copy number of plasmid was increased,and positive single clones were selected for sequencing.Two different plasmid target fragment sequences were obtained.The mutated and unmutated fragments were connected with the original vector by homologous recombination method.Under the same transformation condition and extraction process,the extraction amount of plasmid pUC57 Mu Ori kana was 1.5 times that of original plasmid pUC57 kana.After amplifying culture of plasmid bacteria DH10B pUC57 Mu Ori kana in bioreactor,a good yield was also obtained.

关 键 词：复制起始位点 质粒拷贝数 生物反应器 

分 类 号：R153[医药卫生—营养与食品卫生学]