摘要
猪δ冠状病毒(porcine deltacoronavirus,PDCoV)是近年来新发的一种猪肠道冠状病毒,具有跨物种传播的能力,严重威胁人类和动物健康。为了解PDCoV的致病机制、建立快速检测方法,对PDCoV E蛋白进行原核表达。以PDCoV HNZK-04毒株为模板设计PDCoV E基因的特异性引物,扩增目的基因并克隆至原核表达载体pET-32a中,构建重组表达质粒,转化至大肠杆菌BL21(DE3)诱导表达,并对E蛋白的抗原性进行鉴定。结果显示,成功克隆了大小为252 bp的E基因,构建了重组表达载体pET-32a-PDCoV-E。SDS-PAGE结果表明,成功表达了大小约为30 kDa的重组E蛋白,western blot显示目的蛋白具有良好的反应原性。综上所述,成功构建了PDCoV E蛋白重组质粒并表达了目的蛋白,且重组蛋白具有良好的反应原性,可为PDCoV诊断方法的建立以及进一步研究E蛋白的功能奠定了基础。
Porcine deltacoronavirus(PDCoV)is a newly discovered porcine enteric coronavirus,which has the ability to spread across species and poses a serious threat to human and animal health.In order to understand the pathogenic mechanism of PDCoV and establish a rapid detection method,the prokaryotic expression of PDCoV E protein was performed.PDCoV HNZK-04 strain was used as the template to design the specific primer of PDCoV E gene,amplified the target gene and cloned into the prokaryotic expression vector pET-32a,constructed the recombinant expression plasmid,transformed into E.coli BL21(DE3)to induce expression,and identified the antigenicity of E protein.The results showed that the E gene with a size of 252 bp was successfully cloned and the recombinant expression vector pET-32a-PDCoV-E was constructed.SDS-PAGE results showed that recombinant E protein with a size of about 32 kDa was successfully expressed,and western blot showed that the target protein had good reactivity.In summary,the recombinant plasmid of PDCoV E protein was successfully constructed and expressed the target protein,and the recombinant protein had good regenicity,which could lay a foundation for the establishment of the diagnosis method of PDCoV and the further study of the function of E protein.
作者
田俊娥
党玉丽
TIAN Jun’e;DANG Yvli(Anyang Vocational Secondary School,Anyang Henan 455114;College of Science,Henan Agricultural University,Zhengzhou Henan 450002)
出处
《现代牧业》
2023年第3期18-21,29,共5页
Modern Animal Husbandry
关键词
猪δ冠状病毒
E蛋白
原核表达
Porcine deltacoronavirus
E protein
prokaryotic expression
