利用转座子构建靶向侵袭性抗肿瘤工程菌  

Construction of Invasive Engineered Strain Targeted for Cancer Therapy Using Transposon

作　　者：白利明[1] 唐斯佳 文也[1] 杨琦[1] 孙运军[1] 胡胜标[1] 丁学知[1] 夏立秋[1] 

机构地区：[1]湖南师范大学生命科学学院、微生物分子生物学国家重点实验室培育基地,长沙410081

出　　处：《中国科学：生命科学》2015年第2期200-209,共10页Scientia Sinica(Vitae)

基　　金：国家自然科学基金(批准号:31070006);国家重点基础研究发展计划(批准号:2012CB722301);国家高技术研究发展计划(批准号:2011AA10a203);湖南省高校产业化培育项目(批准号:20134486)资助

摘　　要：转座子是一类在基因组上可以自由跳跃的移动序列,同时也是对微生物进行基因修饰和插入突变的有效工具,但尚未见有利用转座子导入革兰氏阴性菌E.coli Nissle1917菌株的报道.本研究通过构建p R6K转座载体,对肠道益生菌E.coli Nissle1917菌株进行了转座插入诱变,将假结核耶尔森菌的侵袭素基因inv和单核细胞增多性李斯特菌的溶血素基因hly随机整合至E.coli Nissle1917菌株的染色体上,从而使非致病性大肠杆菌E.coli Nissle1917获得侵袭哺乳动物细胞的能力.通过细胞体外侵袭实验发现,本研究所构建的工程菌对B16,HCT-116等肿瘤细胞有较好的侵袭活性,同时与抗肿瘤蛋白Azurin一起作用B16细胞,抗肿瘤效果显著增强,为进一步运用以大肠杆菌E.coli Nissle1917作为DNA疫苗或者基因治疗的载体开辟了新的技术途径.Transposons are a kind of genome sequence jumping freely on the genome sequence and are also valuable tools for gene transfer modification and insertional mutagenesis of microorganisms. However, there has been no report about applications of transposons in gram-negative bacteria E. coli Nisslel917. In this study, we constructed pR6K translocation vector that contains the inv gene from Yersinia pseudotuberculosis and the hly locus from Listeria monocytogenes. Introduction of the inv and hly genes into the chromosomes of E. coli Nisslel917 confers it the ability to invade mammalian cells. Through in vitro experiments, we found that engineered bacteria have good activity for a wide variety of tumor cells, such as B16 and HCT-116. Besides, we transferred Azurin protein expressing vector into the engineered bacteria acting on the B16 cells, greatly improving treatment efficacy. This work has opened up a new way for further application with E. coli Nissle1917 as a carrier of DNA vaccine or gene therapy.

关 键 词：大肠杆菌 转座子 细菌载体 抗肿瘤 

分 类 号：Q78[生物学—分子生物学] R730.54[医药卫生—肿瘤]