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。
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 expressionvector pET-32a, constructed the recombinant plasmid（pET-32a-M）of PDCoV M gene. PET-32a-M recombinant was transformedinto E.coli BL21（DE3）,was expressed by 1 mM concentration of IPTG,further to purify and identify the expressed recombinantPDCoV 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 ofwestern blot showed that the purified recombinant M protein reacted with PDCoV positive serum. This study successfully expressedthe PDCoV M protein, which laid the foundation for the development of PDCoV.
作者
魏姗
石达
陈建飞
冯力
孙东波
Wei Shan;Shi Da;Chen Jianfei;Feng Li;Sun Dongbo(Heilongjiang Bayi Agricultural University,Daqing 163319;Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences)
出处
《黑龙江八一农垦大学学报》
2018年第5期41-45,共5页
journal of heilongjiang bayi agricultural university
基金
国家自然科学基金(31602072)
国家自然科学基金(31572541)
关键词
猪δ冠状病毒
M蛋白
基因表达
蛋白纯化
免疫印迹
porcine δ coronavirus
M protein
gene expression
protein purification
western blot
