单增李斯特氏菌溶血素基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Hemolysin Gene of Listeria monocytogenes

作　　者：吴晓薇[1,2] 徐成刚[1,3] 马保华 江经纬[1] 叶贺佳[1] 区燕宜[1] 徐小芹[1,3] 廖明[1,3] 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]广东检验检疫技术中心,广东广州510623 [3]广东省动物源性人兽共患病预防与控制重点实验室,广东广州510642 [4]南海出入境检验检疫局,广东佛山528200

出　　处：《中国动物检疫》2011年第8期46-50,共5页China Animal Health Inspection

基　　金：广东出入境检验检验局科技项目(2011GDK46)

摘　　要：参考GenBank收录的单增李斯特菌Hly基因序列,设计1对引物,采用PCR技术扩增出单增李斯特氏菌的溶血素基因Hly(不含有信号肽部分),得到一条1590bp的条带。将其连入pMD18-T载体,经酶切、PCR鉴定和序列测定法进行鉴定。测序正确后,将该基因插入到pET-28a中构建原核表达载体pET-28a-sHly,将重组质粒转化到大肠杆菌BL21(DE3),经IPTG诱导,将诱导产物用SDS-PAGE和Western-blot鉴定。结果显示,Hly基因可以在大肠杆菌中获得表达,表达产物分子质量约为65kU,与预期蛋白质分子质量大小一致。经Western-blotting鉴定可知,诱导表达产物以可溶形式存在,可被兔抗LM阳性血清特异识别,具有较好的抗原活性,为进一步研制基于溶解素蛋白的诊断抗原和特异性单克隆抗体,开展LM的致病与免疫机理研究奠定基础。In this study, the Hly gene ofListeria monocytogenes train C53005 was amplified by PCR using designed primers. Then, the gene was cloned into pMD18-T vector, and indentified by digestion with restriction endonuclease, PCR and sequencing. After the sequences were confirmed correct, a prokaryotic expression vector was constructed by inserting the gene into pET-28a vector. The recombinant plasmid pET-28a- silly was transformed into E.coli BL21 （DE3） competent cells, and was induced with 1PTG. The expressed product was analysed by SDS-PAGE and Western-blot. In result, the Hly gene was highly expressed in E.coli and the expressed protein was 65 Ku in size as expected. Western-blot test demonstrated that the expressed protein could specifically react with rabbit anti-LM serum. The results showed the expressed protein was in soluble form with satisfactory antigenicity.

关 键 词：单核细胞增生性李斯特氏菌 溶血素基因 克隆 原核表达 

分 类 号：S852.61[农业科学—基础兽医学]