单增李斯特菌内化素家族蛋白Lmo1136生物学特性的研究  

Study on the biological characteristics on the internalins lmo1136 of Listeria monocytogenes

作　　者：叶泽安 杨立锋[2] 丁强[2] 宋厚辉 孙静 程昌勇 陈绵绵 YE Ze-an;YANG Li-feng;DING Qiang;SONG Hou-hui;SUN Jing;CHENG Chang-yong;CHEN Mian-mian(China-Australia Joint Laboratory for Animal Health Big Data Analytics/Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management/Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics&Advanced Technology/Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province/College of Animal Science and Technology&College of Veterinary Medicine,Hangzhou 311300,China;Ningbo College of Health Sciences,Ningbo 315100,China)

机构地区：[1]浙江农林大学动物科技学院/动物医学院/浙江省畜禽绿色生态健康养殖应用技术研究重点实验室/动物健康互联网检测技术浙江省工程研究中心/浙江省动物医学与健康管理国际科技合作基地/中澳动物健康大数据分析联合实验室,浙江杭州311300 [2]宁波卫生职业技术学院,浙江宁波315100

出　　处：《中国预防兽医学报》2024年第10期997-1004,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划项目(2023YFD1801800);宁波市自然科学基金一般项目(2023J241);浙江省自然科学基金(LY23C180002)。

摘　　要：单增李斯特菌内化素家族蛋白通常在该菌感染及致病中起重要作用,为探究其内化素家族蛋白Lmo1136在李斯特菌感染致病机制中的作用,本研究以单增李斯特菌EGD-e基因组为模板,经PCR扩增lmo1136基因的上下游基因片段,经重叠延伸PCR将同源臂连接后与温敏型穿梭质粒pKSV7连接构建重组质粒,利用同源重组技术使重组质粒与EGD-e同源片段发生交换,得到缺失株Δlmo1136并经PCR和测序鉴定;以单增李斯特菌EGD-e基因组为模板,PCR扩增lmo1136基因与启动子片段,并与整合型穿梭质粒pIMK2连接后电转化Δlmo1136感受态细胞,得到回补株CΔlmo1136并经PCR和测序鉴定。将单增李斯特菌EGD-e、Δlmo1136和CΔlmo1136分别接种96孔板,37℃培养12h后测定各菌株OD600nm值,并根据OD600nm值绘制各菌株的生长曲线。结果显示各菌株生长速度无显著差异(P>0.05)。将各菌株以感染复数(MOI)10感染人结直肠腺癌细胞(Caco-2细胞)后30min和90min时收集细胞裂解物,接种于BHI培养基进行单菌落计数,结果显示,与EGD-e相比,Δlmo1136的黏附性无显著差异(P>0.05),侵袭力显著下降(P<0.05),而CΔlmo1136的黏附性与侵袭力均无显著差异(P>0.05)。将各菌株均以MOI0.2感染小鼠巨噬细胞(RAW246.7细胞)后2h、5h和8h时收集细胞裂解物接种于BHI培养基,单菌落计数,结果显示,与EGD-e相比,Δlmo1136和CΔlmo1136菌落数均无显著差异(P>0.05)。将各菌株均以1×10^(6) cfu/只经腹腔注射小鼠,48 h后取小鼠脾脏与肝脏研磨,接种于BHI培养基进行单菌落计数,结果显示,与EGD-e相比,Δlmo1136的菌落数显著下降(P<0.05),CΔlmo1136的菌落数无显著差异(P>0.05)。将各菌株均以1×10^(6)cfu/只经腹腔注射小鼠,每隔24h记录小鼠存活情况,结果显示,与EGD-e相比,Δlmo1136组小鼠存活率显著升高(P<0.05),CΔlmo1136组小鼠的存活率无显著差异(P>0.05)。上述结果首次表明lmo1136缺失后,单增李斯特菌的生长能力、�Listeria monocytogenes internalins usually play important roles in bacterial infections and its pathogenesis.In order to investigate the role of the internalin family protein Lmo1136 in the pathogenesis of Listeria monocytogenes infection,in this study,Listeria monocytogenes EGD-e genome was used as a template,and the upstream and downstream gene fragments of the lmo1136 gene was amplified by PCR.And a recombinant plasmid was constructed by connecting the homologous arm to the temperature-sensitive shuttle plasmid pKSV7 using overlap extension PCR.The resulting recombinant plasmid was exchanged with the homologous fragment of EGD-e through homologous recombination,and the deletion strainΔlmo1136 was obtained.The Listeria monocytogenes EGD-e genome was used as a template,the lmo1136 gene and promoter fragment were amplified with PCR.The obtained amplicons were ligated with the integrative shuttle plasmid pIMK2 before electrotransferring to the competent cells ofΔlmo1136,and then the complementary strain CΔlmo1136 was obtained.Listeria monocytogenes EGD-e,Δlmo1136 and CΔlmo1136 were inoculated into 96-well plates and cultured at 37℃for consecutive 12 hours.The OD600nm values of each strain were determined,and the results showed that there was no significant difference in the growth rates of the strains(P>0.05).Human colorectal adenocarcinoma cells(Caco-2 cells)was infected with each strain for 30 minutes and 90 minutes at the multiplicity of infection(MOI)10,cell lysis was collected and the lysates were plated on BHI medium for single colony counting.The results showed that compared to EGD-e,there was no significant difference for the adhesion ofΔlmo1136(P>0.05),but there was significantly decreased(P<0.05)for the invasion ofΔlmo1136,in contrast for both the adhesion and invasion of CΔlmo1136,there was no significant difference(P>0.05).The mouse macrophages(RAW246.7 cells)were infected with each strain at an MOI of 0.2 for 2 hours,5 hours and 8 hours,cell lysis was collected and the lysates were plated on

关 键 词：单增李斯特菌 内化素家族蛋白 侵袭力 宿主感染 

分 类 号：S852.6[农业科学—基础兽医学]