金黄色葡萄球菌噬菌体裂解酶LysK的表达及其多克隆抗体的制备  

Expression and pliyclonal antibody preparation of Staphylococcus aureus bacteriophage K lysin LysK

作　　者：李跃[1] 宫鹏娟 夏斐斐[1] 宋军[1] 顾敬敏[1] 张蕾[1] 杨梅[1] 孙长江[1] 冯新[1] 韩文瑜[1] 

机构地区：[1]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医学报》2014年第1期45-49,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基因重点项目(31130072)

摘　　要：将噬菌体与细菌孵育25min提取噬菌体感染细菌的总RNA,通过逆转录的方法获得cDNA,以此为模板进行PCR扩增得到目的片段,以pET-15b为载体构建重组质粒pET-15b-lysK并转入大肠杆菌BL21中,IPTG诱导表达金黄色葡萄球菌噬菌体K裂解酶基因lysK,表达产物蛋白经镍柱纯化后,测定LysK的裂解活性、裂解谱,并免疫家兔制备多克隆抗体。结果表明,成功诱导表达可溶性LysK并获得重组蛋白,酶谱试验证明LysK对金黄色葡萄球菌具有裂解活性,LysK比噬菌体K裂解谱广。制备的兔多克隆抗体能够与LysK发生特异性反应且具有较高效价,为后续进行LysK体内治疗研究打下了基础。The phage were incubated with bacteria for 25 min, then the total RNA including bacteria and bacteriophage was isolated for cDNA Synthesis. Based on a pair of specific primers, the lysK gene was amplified from the cDNA and cloned into the prokaryotic expression vector pET- 15b. This recombinant plasmid pET-15b-lysK was transformed into E. coli BL21. The result showed that the soluble recombinant protein was expressed successfully and had a higher purity using affinity chromatography. The zymogram assay showed that LysK had the killing effects against Staphylococcus aureus. What＇s more, the spectrum of LysK against the Staphylococcus aureus was more broad than bacteriophage K. Finally,the successful preparation of polyclonal anti-bodies laid the foundation for further study.

关 键 词：金黄色葡萄球菌 噬菌体 裂解酶 多克隆抗体 

分 类 号：S852.61[农业科学—基础兽医学]