单增李斯特菌hly基因缺失株的构建及重要特性分析  

Construction and characteristic analysis of Listeria monocytogenes Listeriolysin O encoding gene(hly) deletion strain in mice

作　　者：赵莹莹 贾艳艳[1,2] 杜付玉 余祖华 廖成水[1,2] 何雷 李银聚[1,2] 张春杰 ZHAO Ying-ying;JIA Yan-yan;DU Fu-yu;YU Zu-hua;LIAO Cheng-shui;HE Lei;LI Yin-ju;ZHANG Chun-jie(Luoyang 471000,China;Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control,Luoyang 471000,China)

机构地区：[1]河南科技大学动物疫病与公共卫生重点实验室,河南洛阳471000 [2]洛阳市活载体生物材料与动物疫病防控重点实验室,河南洛阳471000

出　　处：《中国预防兽医学报》2019年第5期509-513,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然基金项目(31702219);河南省自然科学基金(162300410102);河南科技大学省部级科技创新平台培育项目(2015SPT004)

摘　　要：为构建单增李斯特菌(LM)hly基因缺失株,并分析其生物学特性和免疫保护效果,本研究利用同源重组技术获得基因缺失株LM△hly112-153,研究该缺失株对巨噬细胞的侵袭力、溶血能力并测定其LD50,同时评价该缺失株对小鼠的免疫保护效果。结果显示经PCR扩增及序列分析表明获得基因缺失株LM△hly112-153,该缺失株对巨噬细胞的侵袭能力显著下降(p<0.01);其溶血活性比野生株降低2个滴度;并且该缺失株对BALB/c小鼠的LD50为4.253×108 cfu,高于野生株3个数量级;免疫保护试验显示,经缺失株免疫的小鼠对野生株攻毒有75%的免疫保护率。本研究构建了基因缺失株LM△hly112-153,该缺失株对小鼠具有较好的免疫保护效果,以上研究为减毒李斯特菌疫苗载体的研发奠定重要基础。The Listeriolysin strain with O encoding gene(hly)-deletion was constrcted,and its biological characteristics and immunological protection were evaluated in this study.The LM△hly112-153 was obtained by homologous recombination technique.The biological characteristics such as the invasiveness of the deletion strain on macrophages,hemolysis ability and the 50%lethal dose(LD50)were studied,and the protective immunity of LM△hly112-153 in mice was evaluated.The results showed that the hly gene-deletion strain LM△hly112-153 was constructed successfully by the identification of PCR and sequence analysis.The invasive ability of the deletion strain LM△hly112-153 on macrophages is significantly decreased(p<0.01).The hemolytic activity of the gene-deletion strain was reduced by 2 titers compared with the wild strain.And the LD50 of LM△hly112-153 in BALB/c mice was4.253×108 cfu,which was higher than the parent strain by 3 orders of magnitude.Furthermore,in immunological protection experiment,LM△hly112-153 could protect mice against wild-type strain challenge,its protection rate was as high as 75%.The data demonstrate that the hly gene deletion strain LM△hly112-153 constructed in this study has a good protective immunity in mice.Our study may give a basis for the research and development of attenuated Listeria vaccine carrier.

关 键 词：单增李斯特菌 hly基因 安全性 基因缺失 同源重组 

分 类 号：S852.61[农业科学—基础兽医学]