摘要
本研究采用RT-PCR扩增猪δ冠状病毒(PDCoV)的核衣壳蛋白N基因,构建原核表达质粒pET-32a-N,转化至大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达,通过Ni-NTA亲和层析对表达蛋白进行纯化,将纯化后的重组蛋白免疫小鼠,以PDCoV病毒包被酶标板,采用ELISA方法检测小鼠血清抗体,评价重组蛋白的免疫原性。结果显示,PDCoV重组N蛋白(约58 kDa)在0.2 mmol/L IPTG、20℃条件下诱导12 h表达效果最好,且以可溶性形式存在;表达蛋白经Ni柱纯化后能与抗PDCoV阳性血清发生特异性免疫印迹反应,且能够诱导小鼠产生抗PDCoV特异性抗体,具有良好的免疫原性和反应原性。本研究为下一步建立PDCoV检测方法和开展PDCoV N蛋白功能研究奠定了基础。
The N gene of Porcine deltacoronavirus(PDCoV)was amplified by RT-PCR and prokaryotic expression plasmid(pET-32a-N)was constructed followed by transfer into the competent cells of E.coli BL21 and induction by IPTG in the present study.The expressed N protein was purified by Ni-NTA affinity chromatography and used to immunize BALB/c mice.The reactivity of the recombinant N protein was assessed in ELISA using the prepared mouse serum.The results showed that the recombinant N protein with molecular mass of 58 kDa expressed in soluble form with 0.2 mmol/L IPTG at 20℃for 12 h reacted with PDCoV positive serum in ELISA and also induced anti-PDCoV specific antibodies in mice,suggesting it had good immunogenicity and reactivity.This study laid the foundation for further development of PDCoV assay and functional study of the N protein.
作者
庞俊增
袁嘉康
李林岳
秦保亮
李任峰
赵晓会
王自良
PANG Junzeng;YUAN Jiakang;LI Linyue;QIN Baoliang;LI Renfeng;ZHAO Xiaohui;WANG Ziliang(College of Animal Science and Veterinary Medicine,Henan Institute of Science and Technology,Innovative Research Team for Animal Disease Prevention and Control in University of Henan Province,Xinxiang 453003,China;Animal Disease Prevention and Control Center of Xinxiang City,Xinxiang 453003,China;Sanquan College of Xinxiang Medical University,Xinxiang 453000,China)
出处
《中国动物传染病学报》
北大核心
2025年第3期116-122,共7页
Chinese Journal of Animal Infectious Diseases
基金
河南省重点研发与推广专项(科技攻关)-农业领域项目(212102110098,242102111010)。
作者简介
第一作者:庞俊增,男,硕士研究生,兽医专业,主要从事动物传染病学与分子免疫学研究;通信作者:李任峰,E-mail:lirenfeng@sina.com。
