单核细胞增生李斯特菌菌膜形成突变株的筛选  

Screening of Listeria Monocytogenes Mutants on Biofilm Formation

作　　者：马瑜丹[1] 朱欣娜[1] 龙飞[1] 史贤明[1,2] 

机构地区：[1]上海交通大学农业与生物学院食品科学系陆伯勋食品安全研究中心,上海200240 [2]上海食品安全工程技术研究中心,上海201203

出　　处：《中国食品学报》2009年第2期11-17,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：“十一五”国家科技支撑计划课题(2006BAK02A14);上海市科委2007重大科技攻关项目(07dz19508)资助

摘　　要：通过对功能和调控基因的研究,探讨单核细胞增生李斯特菌菌膜的形成机制,建立防治该细菌污染食品的方法。使用电转化的方法,将携带转座子Tn917的质粒pTV1-OK转化到单核细胞增生李斯特菌中,诱导转座,获得1 880个突变株,加上前期构建的突变株,使突变库增加到2 200株。使用96孔细胞培养板对随机挑选的1 000余株突变株进行菌膜培养,结晶紫染色后测OD595值。以此值作为菌膜形成量的筛选依据,最终挑选出菌膜形成能力变小的突变株8株,目标突变获得率约8‰。对筛选的菌膜形成量变小的突变株进行荧光显微观察,证实该8株突变株菌膜形成能力弱于野生菌株。通过PCR验证,这8株突变株的基因组中插入了Tn917,这可能是它们的表型发生变化的原因。Characterization of the functional genes and related regulation elements is the basis to understand the mechanism of bacterial biofilm formation, which is important for the development of prevention methods to the bacterial pollution. The transposon Tn917 was used to produce insertional mutagenesis of L. monoeytogenes by electroporation, and 1880 mutants were obtained in this study, which extended the mutants in our lab to the total amounts of 2200. A thousand of mutants was screened for the negative mutation on biofilm formation by crystal violet staining analysis after microtiter incubation. Eight strains with negative mutation were finally sorted out, and the positive rate of targeted mutation amounts was estimated as 8‰. The biofihn formation ability of these mutants was further confirmed by fluorescence microscopy. It was also confirmed by PCR assay that Tn917 had been inserted into genomes of these targeted mutants. This may be a reason of their phenotypie variation.

关 键 词：单核细胞增生李斯特菌 TN917 菌膜 

分 类 号：R378[医药卫生—病原生物学] R155[医药卫生—基础医学]