SARS-CoV-2 Nsp8基因及蛋白分子特征探讨

张凌寒; 晋乐飞; 陈帅印; 岳利敏; 段广才

doi:10.16462/j.cnki.zhjbkz.2021.04.012

SARS-CoV-2 Nsp8基因及蛋白分子特征探讨

doi: 10.16462/j.cnki.zhjbkz.2021.04.012

1.
450001 郑州，郑州大学第一附属医院生殖医学中心
2.
450001 郑州，郑州大学公共卫生学院流行病学教研室

基金项目:

国家科技重大专项 2018ZX10301407

详细信息

通讯作者:
段广才，E-mail: gcduan@zzu.edu.cn

中图分类号: R373
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- 被引次数: 0
出版历程
- 收稿日期: 2020-08-30
- 修回日期: 2020-11-08
- 网络出版日期: 2021-05-11
- 刊出日期: 2021-04-10

Investigation on molecular characters of SARS-CoV-2 Nsp8 gene and protein

1.
Center for Reproductive Medicine, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450001, China
2.
Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou 450001, China

Funds:

Major National Science and Technology Projects 2018ZX10301407

More Information

Corresponding author: DUAN Guang-cai, E-mail: gcduan@zzu.edu.cn

摘要

摘要: 目的 SARS-CoV-2感染不仅引起呼吸系统病变，且可能累及生殖系统及中枢神经系统。本研究探讨SARS-CoV-2 nsp8基因变异、Nsp8蛋白结构与生物学功能及靶向药物，为建立更有效的防控策略奠定基础。方法采用生物信息学技术和大型生物数据库，分析nsp8基因变异性、Nsp8理化特征、空间结构、抗原表位、生物功能、药物结合靶点等。结果基于3种28株冠状病毒的nsp8序列构建了进化树；SARS-CoV-2毒株间nsp8基因保守性为99%~100%，与SARS遗传距离较MERS近；Nsp8无信号肽及跨膜区；在体外网织红细胞内半衰期为4 h，具有亲水性；获得Nsp8二级和三级结构模型；Nsp8具有线性B细胞和CTL细胞抗原表位、磷酸化及SUMO修饰位点；从DrugBank数据库筛选出可靶向结合Nsp8的四种药物。结论 Nsp8具有典型抗原分子特征，参与病毒复制，且同种不同株nsp8基因高度保守，在病毒致病机制、分型及防治研究中具有应用潜力。在全球亟需新型冠状病毒肺炎特效防治措施的状况下，靶向结合Nsp8的化疗药物具有重要科学意义和应用价值。
- 新型冠状病毒 /
- Nsp8 /
- 靶向药物 /
- 进化树 /
- 抗原表位
Abstract: Objective To explore SARS-CoV-2 nsp8 genetic variation, Nsp8 protein structure, biological function and targeted drugs, and to lay foundation for establishing more effective prevention and control strategies. Methods Analyses of nsp8 genetic variability, physical and chemical characteristics, spatial structure, antigenic epitopes, biological function, and drug combined targets of Nsp8 were carried out using bioinformatics technology and large biological databases. Results Based on nsp8 sequences of 28 isolates of coronavirus of three species, evolutionary tree was successfully constructed. SARS-CoV-2 isolates showed 99%-100% conservation of nsp8 genes, less genetic distance to SARS than MERS isolates. Nsp8 had no signal peptide and transmembrane area. In reticulocytes in vitro, Nsp8 had a half-life of 4 h and was hydrophilic. A secondary model and a tertiary structure model were established. Linear B cell and CTL antigenic epitopes, phosphorylation and SUMB modification sites were found in Nsp8. Using the DrugBank database, four drugs targeted Nsp8 were obtained. Conclusions Nsp8 possesses the characteristics of typical antigens, participates in viral replication, and various isolates of the same species share high conservation of nsp8 gene, suggesting potential applications in researches on pathogenic mechanism, genotyping and prevention of this virus. Notably, this is the first report on Nsp8-targeted chemotherapeutic drugs, and the findings can be of considerable scientific significance and application value, under the conditions that measures with special effect for COVID-19 prevention and control are urgently needed.
- Severe acute respiratory syndrome coronavirus 2 /
- Nsp8 /
- Targeted drugs /
- Evolutionary tree /
- Antigenic epitope

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图 1 SARS-CoV-2、SARS及MERS病毒株nsp8基因遗传进化树

Figure 1. Genetic evolutionary tree of nsp8 genes of SARS-CoV-2, SARS and MERS isolates

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图 2 Nsp8亲疏水性分析

Figure 2. Hydrophilicity/hydrophobicity analysis of Nsp8

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图 3 Nsp8信号肽分析

Figure 3. Signal peptide analysis of Nsp8

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图 4 Nsp8跨膜区分析

Figure 4. Analysis of Nsp8 transmembrane region

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图 5 Nsp8磷酸化分析

Figure 5. Nsp8 phosphorylation analysis

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图 6 Nsp8的SUMOplot分析

Figure 6. SUMOplot analysis of Nsp8

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图 7 Nsp8保守结构域预测

Figure 7. Prediction of Nsp8 conserved junction

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图 8 Nsp8二级结构预测

Figure 8. Secondary structure prediction of Nsp8

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图 9 Nsp8三级结构的SWISS-MODEL分析

Figure 9. Analysis of tertiary structure of Nsp8 using SWISS-MODEL

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图 10 Nsp8 B细胞线性抗原表位分析

Figure 10. Analysis of B-cell antigenic epitopes of Nsp8

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图 11 基于DrugBank的Nsp8靶向结合药物分子分析

注：A：4-(Dimethylamino) benzoic acid, C9H11NO2, DB08748；B：S-[5-(TRIFLUOROMETHYL)-4H-1, 2, 4-TRIAZOL-3-YL] 5-(PHENYLETHYNYL) FURAN-2-CARBOTHIOATE, C16H8F3N3O2S, DB07743；C：2-[(2, 4-DICHLORO-5-METHYLPHENYL)SULFONYL]-1, 3 -DINITRO-5- (TRIFLUOROMETHYL) BENZENE, C14H7Cl2F3N2O6S, DB07620; D：NALPHA-[(BENZYLOXY)CARBONYL]-N-[(1R)-4-HYDROXY-1-METHYL-2-OXOBUTYL] -L-PHENYLALANINAMIDE, C22H26N2O5, DB08732。

Figure 11. Analysis of Nsp8-targeted drugs based on DrugBank

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表 1 冠状病毒SARS-COV-2、SARS和MERS毒株Nsp8比较

Table 1. Comparison of Nsp8 proteins of coronavirus SARS-COV-2, SARS and MERS strains

属性	病毒株
属性	SARS-COV-2/CHN/ Wuhan-Hu-1/2020	SARS/CHN/GD01/2003	MERS/KOR/066-2015/2015
基因结构	nsp8基因为多聚蛋白基因ORF1ab中一段编码区，长594 bp，GC含量38.38%	nsp8基因为多聚蛋白基因ORF1ab中一段编码区，长594bp，GC含量42.08%	nsp8基因为多聚蛋白基因ORF1ab中一段编码区，长597 bp，GC含量41.71%
分子质量(Dal)	21 881.08	21 866.03	21 886.17
氨基酸数量(aa)	198	198	199
等电点	6.58	6.58	6.83
半衰期(h)	4.4	4.4	4.4
不稳定系数	37.78	39.23	25.77
脂肪族指数	88.33	87.83	95.63
平均亲水系数	-0.192	-0.226	-0.101
Nsp8二级结构	α螺旋54.04%、随机卷曲22.22%、β转角6.57%和延伸链17.17%	α螺旋52.02%、随机卷曲21.21%、β转角6.06%和延伸链20.71%	α螺旋53.27%、随机卷曲23.12%、β转角6.03%和延伸链17.59%
Nsp8三级结构
磷酸化位点	15个丝氨酸、13个苏氨酸和6个酪氨酸磷酸化位点	14个丝氨酸、12个苏氨酸和7个酪氨酸磷酸化位点	15个丝氨酸、11个苏氨酸和5个酪氨酸磷酸化位点
糖基化位点	无O-糖基化位点	3个O-糖基化位点	无O-糖基化位点
B细胞抗原表位(长度≥9 aa)	4个B细胞表位：23~33、50~58、73~81、158~179 aa	4个B细胞表位：23~33、50~58、73~81、158~178 aa	4个B细胞表位：23~34、50~59、74~82、159~179 aa
HLA-A*02∶01限制性CTL细胞表位(长度=9 aa，分值≥20)	14个CTL表位：90~98、152~160、1~9、25~33、34~42、102~110、151~159、180~188、54~62、123~131、179~187、86~94、118~126、161~169 aa。	与SARS-COV-2/CHN/ Wuhan-Hu-1毒株相同	6个CTL表位：91~99、103~111、115~123、35~43、38~46、159~167 aa
Nsp8靶向结合药物	DrugBank Accession No. DB08748、DB07620、DB07743、DB08732	与SARS-COV-2/CHN/ Wuhan-Hu-1毒株相同

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