重组单增李斯特菌溶血素O在大肠杆菌中的分泌表达及活性分析  

Secretory expression of recombinant Listeria monocytogenes listeriolysin O in E.coli and analysis of its biological activity

作　　者：李文艳[1,2] 张永红[1] 林洪羽[3] 李秀锦[3] 仲飞[1] 

机构地区：[1]河北农业大学动物医学院 河北省兽用生物制品工程技术研究中心,河北保定071001 [2]河北大学基础医学院,河北保定071001 [3]燕山大学环境与化学工程学院,河北秦皇岛066004

出　　处：《中国兽医学报》2014年第8期1278-1283,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31140093);河北省自然科学基金资助项目(C2013204130)

摘　　要：单增李斯特菌是食源性致病菌,常引起人和动物的生殖障碍,由此菌分泌的溶血素O(LLO)是重要的致病因子。为研究LLO对细胞的毒性作用及对动物体生殖免疫的调节作用,需要制备大量重组LLO。为此本试验依据LLO基因序列(JN703918)通过PCR方法从单增李斯特菌菌株基因组中扩增无信号肽的LLO编码基因,然后将其插入到pET-20b(+)质粒中,使其5′端与载体中的pelB信号肽序列融合,3′端与His-标签融合,构建成LLO分泌性原核表达载体pET-20b-LLO/H。将载体分别转化大肠杆菌BL21(DE3)pLysS,在IPTG的诱导下进行表达,利用Ni-NTA琼脂糖凝胶从细菌周质释放液中提取和纯化重组LLO蛋白,并通过SDS-PAGE及Western blot进行鉴定。利用溶血试验检测重组蛋白的生物活性。结果表明,本试验扩增的LLO基因序列与GenBank中的LLO基因序列完全一致。采用分泌性表达策略实现了重组LLO在大肠杆菌中的分泌表达,并且表达的重组LLO具有明显的溶血活性。Listeria monocytogenes is a food-born pathogen and often causes human and animal re productive disorder. Its secretory product, listeriolysin O （LLO）, is considered as an important pathogenic factor. To study its toxic effects on the host ceils and its regulatory function to the reproductive immunity of animals,the plenty of the recombinant LLO are needed. Therefore,in this study,the LLO gene without signal peptide was amplified by PCR using the specific primers designed based on LLO gene sequence（GenBank：JNT03918） and inserted into pET-20b（＋） plasmid to construct the prokaryotic secretory expression vector pET-20b-LLO/H,in which the pelB signal peptide and His-tag were fused at 5＇and 3＇terminus of LLO gene,respectively. The pET-20b-LLO/ H vector was transformed into E. coli BL21（DE3） pLysS and the recombinant LLO was expressed under the IPTG induction. The expressed LLO was released from bacteria periplasm by MgSO4 shock and purified by Ni-NTA agarose beads. The LLO was identified with SDS-PAGE and Western blot and its biological activity was determined using the method of hemolysis assay. The results showed the sequence of LLO gene amplified in this study consisted with that in GenBank. The recombinant LLO was successfully expressed in a secretory manner in E. coli. The expressed LLO was identified to possess the hemolysis activity, indicating that the recombinant LLO has the biological activity.

关 键 词：溶血素O 单增李斯特菌 大肠杆菌 分泌表达 生物活性 

分 类 号：S852.61[农业科学—基础兽医学] S855.99[农业科学—兽医学]