食源性单增李斯特菌毒力岛4基因缺失株的构建及其体外毒力研究  

Construction of A Food-Borne Listeria monocytogenes Virulence Island 4 Gene Deletion Strain and Its in vitro Virulence Study

作　　者：阮婷玉 康立超[2] 刘扬扬 李红欢 李楠 马勋[1] 王静[1] RUAN Tingyu;KANG Lichao;LIU Yangyang;LI Honghuan;LI Nan;MA Xun;WANG Jing(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;Analysis and Testing Center,Xinjiang Academy of Agricultural and Reclamation Science,Shihezi 832000,China)

机构地区：[1]石河子大学动物科技学院,石河子832003 [2]新疆农垦科学院分析测试中心,石河子832000

出　　处：《中国动物传染病学报》2023年第2期36-43,共8页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金项目(31860712);石河子大学高层次人才科研启动项目(RCZX201504);石河子大学应用基础研究青年项目(2015ZRKXYQ02);省部共建绵羊遗传改良与健康养殖国家重点实验室开放课题(MYSKLKF20190)。

摘　　要：为研究单增李斯特菌毒力岛4(LIPI-4)对单增李斯特菌(LM)LM928毒力强弱的影响及其在LM穿越血脑屏障时发挥的作用。本研究利用同源重组技术构建LIPI-4缺失株,将亲本株与缺失株以不同的MOI值感染脑微血管内皮细胞(HCMEC)后,通过细胞增殖与细胞毒性试剂盒(CCK-8)检测细胞活性,空斑试验检测细胞病变及细菌胞间迁移率以及胞内增殖试验检测LIPI-4缺失后对LM胞内增殖的影响。结果表明:本试验成功构建LIPI-4缺失株。与亲本株相比,LIPI-4缺失株侵染HCMEC后,细胞活性极显著增强(P<0.01);空斑形成数量极显著降低(P<0.01);胞内增殖速度减慢,胞内增殖12 h时菌量极显著减少(P<0.01)。体外细胞实验表明LIPI-4的缺失显著降低了LM的毒力。研究结果可为LIPI-4在LM穿越血脑屏障时的作用机制提供研究基础。To investigate the effect of Listeria monocytogenes pathogenicity island 4(LIPI-4)on the virulence and its role in the blood-brain barrier,LIPI-4 deletion strain was constructed by homologous recombination technique.The parental strain and deletion strain were inoculated into brain microvascular endothelial cells(HCMEC)at different MOIs.Then the cell viability was examined by cell proliferation and cytotoxicity kit,the cytopathic effect and bacterial intercellular mobility by plaque assay and the effect of LIPI-4 deletion on LM intracellular proliferation by intracellular proliferation assay.The results showed that the LIPI-4 deletion strain was successfully constructed in this experiment.As compared with the parental strain,LIPI-4 deletion strain infection caused significant enhancement(P<0.01)of the activity of HCMEC,significant decrease(P<0.01)of the number of plaques,slow down of intracellular proliferation rate and significant reduction(P<0.01)of the number of bacteria at 12 hours of intracellular proliferation.In vitro cell experiments indicated that deletion of LIPI-4 significantly reduced the virulence of LM.These results provided a research basis for the mechanism of action of LIPI-4 when LM crossed the blood-brain barrier.

关 键 词：食源性单增李斯特菌 LIPI-4 血脑屏障 毒力 

分 类 号：S852.61[农业科学—基础兽医学]