摘要
为建立一种有效提取高纯度副猪嗜血杆菌(Haemophilus parasuis,HPS)脂多糖(Lipopolysaccharides,LPS)的方法,进一步阐明HPS的致病机制,通过热酚水法提取LPS,浓缩后,采用酶解法和超离法纯化提取物,分别用硫酸-苯酚法、BCA法、紫外分光光度法,测定LPS提取物中多糖、蛋白及核酸的含量,通过SDSPAGE电泳银染方法鉴定提取物。将提取的LPS作用于猪肺泡巨噬细胞,通过荧光定量方法检测IL-1β的表达量差异。结果显示:纯化的LPS平均产率为2.56%;LPS提取物中不含还原性单糖,多糖含量为3.27%,蛋白含量为0.77%,核酸含量为0.90%;提取的LPS能够引起猪肺泡巨噬细胞IL-1β表达量显著上调。因此,通过该方法提取纯化的LPS可以用于HPS与宿主细胞间作用机制的研究。
In order to establish a method for effectively extracting high-purity lipopolysaccharide（LPS）from Haemophilus parasuis（HPS）,and lay a foundation for clarifying the pathogenesis of HPS. Firstly,the LPS was extracted by hot phenol. Then the LPS extracts were purified by enzymolysis and ultracentrifugation after concentration. The contents of polysaccharides,proteins and nucleic acids in LPS extracts were determined by sulfuric acid-phenol,BCA and ultraviolet spectrophotometric methods. The extracts were identified by SDS-PAGE electrophoresis silver staining. At last,porcine alveolar macrophages were infected with LPS extracts,and the difference of IL-1β expression was detected by fiuorescence quantitative PCR. The results showed that the average yield rate of purified LPS was 2.56%,and LPS extracts didn＇t contain the reducing monosaccharide. The contents of polysaccharides,proteins,nucleic acids were 3.27%,0.77% and 0.90%,respectively. Moreover,the expression of IL-1β in porcine alveolar macrophages was significantly up-regulated after LPS treatment. Therefore,the purified LPS by this method could be used for studying the interaction mechanism between HPS and host cells.
作者
杨杰
李建达
丛晓燕
杨胜男
任素芳
郭立辉
陈智
陈蕾
曾昊
孙文博
时建立
李俊
邱文彬
吴家强
于江
Yang Jie;Li Jianda;Cong Xiaoyan;Yang Shengnan;Ren Sufang;Guo Lihui;Chen Zhi;Chen Lei;Zeng Hao;Sun Wenbo;Shi Jianli;Li Jun;Qiu Wenbin;Wu Jiaqiang;Yu Jiang(Institute of Animal Science and Veterinary Medicine,Shandong Academy of Agricultural Sciences,Key Laboratory of Animal Disease Control and Breeding of Shandong Province,Jinan,Shandong250100,China;Qingdao Animal Disease Prevention and Control Center,Qingdao,Shandong266000,China;College of Life Sciences,Shandong Normal University,Jinan,Shandong250014,China)
出处
《中国动物检疫》
CAS
2018年第9期106-111,共6页
China Animal Health Inspection
基金
国家自然科学基金(31600106)
国家重点研发计划项目(2017YFD0500602)
山东省自然科学基金(ZR2015YL078
ZR2014YL013)
关键词
副猪嗜血杆菌
脂多糖
提取
鉴定
Haemophilus parasuis
lipopolysaccharide
extraction
identification
