【摘要】 δ冠状病毒是一种可以感染哺乳动物和鸟类的新型冠状病毒,其中,猪δ冠状病毒可以引发仔猪的呕吐、腹泻、脱水,甚至死亡,给养猪业造成了巨大的损失。为了获得猪δ冠状病毒(Porcine deltacoronavirus,PDCoV)M基因重组蛋白,试验克隆了PDCoV NH毒株的M基因,并连接至pET-32a原核表达载体,构建了PDCoV M基因重组质粒(pET-32a-M),将pET-32aM重组质粒转化到大肠杆菌BL21(DE3)后,利用1 mM浓度IPTG进行诱导表达,进一步对表达的PDCoV重组M蛋白进行纯化与鉴定。结果显示,以PDCoV NH毒株病毒RNA为模板,通过RT-PCR扩增出约654 bp的M基因,与预期大小相符;SDSPAGE电泳结果显示,PDCoV M基因在大肠杆菌BL21(DE3)中成功获得表达,表达的重组M蛋白大小为42 kD,与预期大小相符;通过western blot进行鉴定,结果显示,纯化的重组M蛋白能与PDCoV阳性血清发生反应,具有良好的抗原性。研究成功表达了PDCoV M蛋白,为PDCoV的进一步研究奠定了基础。更多还原

【Abstract】 δ coronavirus was a novel coronavirus that could infect mammals and birds. Porcine δ coronavirus could cause vomiting,diarrhea,dehydration and even death of piglets,causing great losses to the pig industry. In order to obtain the porcine δ coronavirus(PDCoV)M gene recombinant protein,the M gene was cloned by PDCoV NH strain and connected to the prokaryotic expression vector pET-32 a,constructed the recombinant plasmid(pET-32 a-M)of PDCoV M gene. PET-32 a-M recombinant was transformed into E.coli BL21(DE3),was expressed by 1 mM concentration of IPTG,further to purify and identify the expressed recombinant PDCoV M protein. The results showed that the PDCoV virus NH strain RNA as template,M gene was amplified by RT-PCR about654 bp,consistent with the expected size;the results of SDS-PAGE showed that the PDCoV M gene in Escherichia coli BL21(DE3)was successfully expressed,the expression of the recombinant M protein was 42 kD,consistent with the expected size;the results of western blot showed that the purified recombinant M protein reacted with PDCoV positive serum. This study successfully expressed the PDCoV M protein,which laid the foundation for the development of PDCoV.更多还原