摘要:
根据鸭新城疫病毒NP基因序列设计1对特异性引物,RT-PCR方法扩增出NP基因,克隆入原核表达载体pET-30a,转化大肠杆菌感受态细胞BL21(DE3),在IPTG的诱导下融合蛋白获得了成功表达,SDSPAGE结果显示表达的融合蛋白分子量约为59kD,Western blotting分析表明该重组融合蛋白能与His组氨酸单抗发生特异性反应。将目的蛋白切胶免疫ICR小鼠,制备了针对重组蛋白的多抗血清,Western-blotting分析显示制备的多抗血清能够与鸭新城疫病毒感染的SPF鸡胚尿囊液总蛋白发生特异反应。以上结果表明,NP基因在大肠杆菌中获得了成功表达,且制备的多抗血清可以用于NP蛋白的检测。
Abstract:
According to the genome sequences of the duck NDV,apair of specific primers was designed.The NP gene of the virus was amplified by RT-PCR and cloned into the prokaryotic expression vector pET-30 a.The recombinant plasmids were transformed into BL21(DE3)E.coli.Subsequently,this recombinant fusion protein was successfully expressed following IPTG induction.SDS-PAGE showed that the protein had an approximate molecular weight of 59 kD,and the Western blotting assay revealed that it could be recognized by the monoclonal antibody against histidine-tagged protein.The antiserum was,then,produced by the immunized ICR mice with the recombinant protein.Western blotting on the antiserum indicated its specific reactivity with NDV allantoic fluid.It was concluded that the NP gene could be successfully expressed in E.coli,and the antiserum could be used for detection of NPprotein.
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