单增李斯特菌膜裂解相关基因缺失突变株的构建及生物学特性鉴定

doi:10.11843/j.issn.0366-6964.2016.04.018

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (4): 779-788.doi: 10.11843/j.issn.0366-6964.2016.04.018

单增李斯特菌膜裂解相关基因缺失突变株的构建及生物学特性鉴定

江玲丽¹，高有领²，周向阳³，白帆⁴，方春⁴，钱国英^2*，方维焕⁴

（1.宁波卫生职业技术学院，宁波 315100；2.浙江万里学院，宁波 315100；3.舟山出入境检验检疫局，舟山 316000；4.浙江大学动物预防医学研究所，杭州 310058）

收稿日期:2015-10-10 出版日期:2016-04-23 发布日期:2016-04-26
通讯作者: 钱国英，教授，E-mail：qiangy@zwu.edu.cn
作者简介:江玲丽（1980-），女，浙江温岭人，研究员，博士，主要从事微生物与食品安全研究，Tel：0574-88126061，E-mail：jllgrace@163.com
基金资助:
浙江省科技厅公益技术研究农业项目(2014C32047)；2015年度留学人员科技活动项目择优资助经费项目；浙江万里学院省重中之重学科开放基金（KF2014007）；宁波市自然科学基金（2015A610189）；浙江检验检疫局重点科研项目（2014-ZKZ-017）

Construction and Characterization of Mutant Strains from Listeria monocytogenes with Deletion of Vacuolar Lysis Related Genes

JIANG Ling-li¹，GAO You-ling²，ZHOU Xiang-yang³，BAI Fan⁴，FANG Chun⁴，QIAN Guo-ying^2*，FANG Wei-huan⁴

(1.Ningbo College of Health Sciences，Ningbo 315100，China；2.Zhejiang Wanli University，Ningbo 315100，China；3.Zhoushan Entry-Exit Inspection and Quarantine Bureau，Zhoushan 316000，China；4.Institute of Preventive Veterinary Medicine，Zhejiang University，Hangzhou 310058，China)

Received:2015-10-10 Online:2016-04-23 Published:2016-04-26

摘要/Abstract

摘要：

拟探明单核细胞增多性李斯特菌（单增李斯特菌）分离株M7可能的低致病力机制。利用SOE-PCR及同源重组法构建M7膜裂解相关基因hly及plcB单缺失（M7-Δhly和M7-ΔplcB）及双缺失突变株（M7-Δhly/plcB），比较它们与亲本株的生物学特性差异。结果如下：PCR及反转录PCR表明突变株构建成功。突变株M7-Δhly和M7-Δhly/plcB因hly缺失致其溶血活性丧失。plcB缺失突变株M7-ΔplcB和M7-Δhly/plcB无可见溶脂活性。单缺失突变株M7-ΔplcB、M7-Δhly与M7具有相似的细胞黏附及增殖能力（P＞0.05）。MOI为1 000时，双缺失突变株M7-Δhly/plcB对Caco-2细胞毒性最低（11.10%），M7-Δhly次之（23.53%）（P<0.05）。空斑试验表明菌株M7和M7-ΔplcB仅能在无庆大霉素培养基中形成空斑。hly及plcB缺失致单增李斯特菌对免疫抑制小鼠毒力降低。单增李斯特菌M7低致病力可能与hly及plcB的高水平表达有关，致细菌对宿主细胞毒性增强而从细胞内逸出，使其不能躲避宿主免疫系统而被清除。

关键词: 单增李斯特菌, 膜裂解相关基因, 细胞毒性, 生物学特性

Abstract:

We attempted to gain insights into the possible mechanism of one Listeria monocytogenes strain M7 with naturally low virulence.Splicing by overlap extension-PCR and homologous recombination were utilized to construct the mutant strains with full deletion of vacuolar lysis related genes hly or (and) plcB.Subsequently，comparisons among mutants and their parent strain M7 together with the reference strain EGD were investigated in terms of hemolytic activity，phospholipase activity，adhesion and intracellular growth in HeLa cells，plaque forming assay in L929 cells，cytotoxicity against Caco-2 cells，as well as virulence to immunocompromised ICR mice.PCR amplification and reverse transcriptional PCR of the target genes indicated the successful construction of the mutants M7-Δhly，M7-ΔplcB and M7-Δhly/plcB.Furthermore，hemolytic and phospholipase activity were devoid in mutants M7-Δhly and M7-ΔplcB，respectively.Neither hemolytic nor phospholipase activity could be seen in the double mutant M7-Δhly/plcB.Besides，mutants M7-Δhly and M7-ΔplcB exhibited similar adhesiveness and intracellular growth in vitro (P＞0.05).Cytotoxicity assay under multiplicity of infection (MOI) ratio 1 000 revealed that the double mutant M7-Δhly/plcB showed the lowest cytotoxic to the host Caco-2 cells with the ratio of 11.10%，followed by mutant M7-Δhly with the ratio of 23.53%.Significant difference could be found between the aforementioned two mutants and other test strains (P<0.05).Plaque forming assay pinpointed that mutant M7-ΔplcB together with its parent strain M7 could form plaques only in the medium without gentamycin.The full deletion of hly or (and) plcB resulted in even less pathogenicity in immunocompromised ICR mice.This study presents some clues as the low virulence of strain M7 probably result from the over-expression of genes hly and plcB.Thereafter，the M7 strain might be exposed to the host immune responses due to its detrimentally stronger cytotoxicity.

Key words: Listeria monocytogenes, vacuolar lysis related genes, cytotoxicity, biological characteristics

中图分类号:

R378.99.4

江玲丽，高有领，周向阳，白帆，方春，钱国英，方维焕. 单增李斯特菌膜裂解相关基因缺失突变株的构建及生物学特性鉴定[J]. 畜牧兽医学报, 2016, 47(4): 779-788.

JIANG Ling-li，GAO You-ling，ZHOU Xiang-yang，BAI Fan，FANG Chun，QIAN Guo-ying，FANG Wei-huan. Construction and Characterization of Mutant Strains from Listeria monocytogenes with Deletion of Vacuolar Lysis Related Genes[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(4): 779-788.

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单增李斯特菌膜裂解相关基因缺失突变株的构建及生物学特性鉴定

Construction and Characterization of Mutant Strains from Listeria monocytogenes with Deletion of Vacuolar Lysis Related Genes

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