作　　者： ; ; ; ; ; ; ; (马保华）;

机构地区： 华南农业大学兽医学院

出　　处： 《中国动物检疫》 2011年第8期46-50,共5页

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

领　　域： [农业科学] [农业科学] [农业科学]