摘要
采用生物信息学方法对新型冠状病毒(SARS-CoV-2)核衣壳蛋白(N蛋白)进行亲疏水性、抗原表位预测及多序列对比分析,构建重组质粒pET28a/N,在大肠杆菌原核表达体系下通过调整诱导温度和时间,提升蛋白溶解度和表达量,并对表达出的重组N蛋白进行纯化和鉴定。结果表明:SARS-CoV-2 N蛋白编码419个氨基酸,等电点(PI)为10.10,无跨膜区,无信号肽序列,局部亲水性较强,全长蛋白抗原指数较高,高度保守,与SARS冠状病毒(SARS-CoV)N蛋白同源性为90.5%;采用大肠杆菌原核表达体系发酵,工程菌BL21(DE3)/pET28a/N在IPTG终浓度为0.2 mmol/L、16℃低温条件下诱导20 h,蛋白呈可溶性表达,且此时蛋白的表达量最高,占总蛋白表达量的70%;通过Ni-NTA亲和层析、凝胶过滤层析纯化后的目的蛋白纯度达90%,分子质量为55 kDa,具有特异性。
Bioinformatics methods were used to predict the hydrophilicity,hydrophobicity,antigen epitopes and analyse multiple sequence alignment of the nucleocapsid protein(N protein)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).The recombinant plasmid pET28a/N was constructed.In the prokaryotic expression system of Escherichia coli,the solubility and expression level of the protein were improved by adjusting the change of induction temperature and time,and the expressed recombinant N protein was purified and identified.The results showed that SARS-CoV-2 N encoded 419 amino acids,with an isoelectric point(PI)of 10.10,no transmembrane region,no signal peptide sequence,and strong local hydrophilicity.The full-length protein had a high antigenic index and was highly conserved,and its homology with SARS-CoV N protein was 90.5%.After fermentation with Escherichia coli prokaryotic expression system,the engineering strain BL21(DE3)/pET28a/N was induced at 16℃for 20 h with the final IPTG concentration of 0.2 mmol/L,and the protein was soluble and most pressed at this time,accounting for 70%of the total protein expression.The target protein purified by Ni-NTA affinity chromatography and gel filtration chromatography had a purity of 90%and a molecular weight of 55 kDa,which was specific.
作者
樊志浩
李玉林
张恒
王旭东
王云龙
FAN Zhihao;LI Yulin;ZHANG Heng;WANG Xudong;WANG Yunlong(School of Life Sciences,Zhengzhou University,Zhengzhou 450001,China;He′nan Bioengineering Technology Research Center,Zhengzhou 450121,China;He′nan General Hospital,Zhengzhou 450002,China;Department of Bioengineering,Zhengzhou Technical College,Zhengzhou 450121,China)
出处
《轻工学报》
北大核心
2022年第4期34-40,共7页
Journal of Light Industry
基金
河南省新型冠状病毒防控应急科研攻关项目(201100310300)。
