摘要
利用PCR技术从猪细小病毒的DNA模板中扩增了NS1的基因片段,将PCR产物克隆至pET32c载体,将构建好的重组质粒pET32c-NS1,转入表达宿主菌BL21中,利用低温诱导表达的方法,避免了包涵体的形成。Western-blot结果显示,表达产物有良好的生物活性。纯化后的重组蛋白作为抗原,包被酶标板,建立了检测猪细小病毒特异性抗体的间接ELISA方法。抗原最佳包被质量浓度为2.5 mg/L、血清最佳稀释度为1∶200。表达的NS1蛋白可作为免疫诊断试剂用于检测PPV,且能很好的区别灭活疫苗免疫猪和自然感染猪。
The completed NS1 gene was amplified from PPV DNA by PCR method. Then the products were cloned into pET32c vector and the sequence was determined. The recombinant plasrnid pET32c-NSlwas transformed into BL21 competent cells and expressed in super-high level by using low-temperature induction to reduce formation of inclusion body. Western-blot analysis proved the renaturation protein has a good immunoreactivity against PPV antibody. The indirect ELISA method for the detection of PPV specific antibody in procine serum was established,after the optional working circumstances for the ELISA assay （ antigen concentration： 2.5 mg/L;optimal serum dilution： 1 ： 200） with chessboard titration. The recombinant NS1 can be applied in differential diagnosis of PPV infections.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2009年第10期1247-1250,共4页
Chinese Journal of Veterinary Science
基金
上海市农业科学院青年基金资助项目(200503)
