摘要
根据LMO溶血素基因hlyA设计引物,PCR扩增hlyA,将扩增产物与pMD18-T连接,重组质粒经酶切鉴定、PCR分析以及确证性测序。将由重组质粒pMD18-hlyA上扩增的缺失信号肽序列的目的片段与表达载体DGEX-4T-1分别酶切连接后,转化BL21细胞。筛选阳性克隆,用IPTG诱导表达,SDS-PAGE和Western blot分析,纯化重组融合蛋白进行溶血试验、小鼠致病力试验和间接ELISA分析。结果表明hlyA基因在大肠杆菌中成功表达分子量82Ku的融合蛋白,能裂解红细胞,且能被LMO阳性血清所识别。一定剂量腹腔注射能将小鼠致死。以此为包被抗原的间接ELISA可以将LMO阴、阳性血清分开。这表明GST-LLO融合蛋白具有良好的生物活性,可用于李氏杆菌病的间接ELISA诊断。
The hlyA of Listeria monocytogenes was amplified by PCR and ligated with pMD18-T vector. The recombinant plasmid obtained was certified by PCR, restriction endonucleases analysis and sequencing. A prokaryotic expression plasmid, named pGEX-hlyA was constructed subsequently. After induced by IPTG, the expression product was tested by SDS-PAGE and Westem blot. The result showed that 82 Ku fusion protein was recognized by positive serum against Listeria monocytogenes, and the protein could lyse swine red cell. The above results suggest that the Listeria monocytogenes hlyA was successfully expressed in E.coli, providing a solid foundation to develop indirect ELISA for diagnosis of this disease.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2007年第11期852-855,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
中国农业科学院兰州兽医研究所所长基金项目
关键词
产单核细胞李氏杆菌
表达
活性分析
Listeria monocytogenes hlyA
expression
bioactivity analysis
