| 290 | 2 | 269 |
| 下载次数 | 被引频次 | 阅读次数 |
本研究旨在制备人GⅡ.14诺如病毒的（Norovirus, NoV）病毒样颗粒（Virus-like particle, VLP），探索其抗原性与组织血型抗原结合的特征。利用杆状病毒系统表达人GⅡ.14 NoV VP1蛋白，通过超速离心和分子筛层析纯化VLP；将VLP免疫动物制备兔多抗血清，通过免疫印迹（Western blot, WB）、酶联免疫吸附（ELISA）进行抗原性评价及多抗功能验证；利用唾液结合实验检测人GⅡ.14 VLP的糖结合特征；通过蛋白序列及结构比较分析GⅡ.14P蛋白潜在的糖结合位。纯化获得人GⅡ.14 VP1蛋白，电镜观察显示可以形成结构较为均一的VLP；制备的兔多抗能与GⅡ.14 VLP特异性结合，与检测的其他基因型VLP没有交叉反应；唾液结合实验表明GⅡ.14 VLP与人A/B/O/AB型别唾液都有较好的结合；序列和结构分析显示GⅡ.14 P蛋白模拟结构与GⅡ.9最为接近，具有与GⅡ.9以及流行株GⅡ.4等相似的潜在糖结合区。本研究表明GⅡ.14具有较为广泛的唾液结合特性，通过序列比对和结构学方法分析了GⅡ.14潜在的受体结合机制，为不同型别NoVs的流行监测提供了更多依据。
Abstract：This study aimed to get the virus-like particles(VLPs) of human GⅡ. 14 norovirus(NoV) and explore its binding characteristics with histo-blood group antigens(HBGAs). The VP1 protein of human GⅡ.14NoV was expreesed by baculovirus system. VLPs were purified by ultracentrifugation and gel-filtration chromatography. Rabbit polyclonal antisera were generated by immunizing animals with GⅡ. 14 VLPs. The function of polyclonal antibodies was verified by western blotting and enzyme-linked immunosorbent assay. The saliva-binding assay was carried out to detect the glycan-binding characteristics of human GⅡ. 14 VLPs.Potential glycan binding sites of the GⅡ. 14 P protein were analyzed by sequence alignment and structural comparison. The purified GⅡ. 14 VP1 protein formed relatively uniform VLPs when observed by electron microscopy. Rabbit polyclonal antibodies could specifically bind to GⅡ.14 VLPs with no obvious cross-reactivity with other tested VLPs. GⅡ.14 VLPs bound well to human saliva of A/B/O/AB types according to the salivabinding assay. Sequence and structural analyses showed that the simulated structure of the GⅡ.14 P protein was closest to GⅡ.9 P protein, and possessed similar potential glycan-binding sites to that of GⅡ.9 and the epidemic GⅡ.4. Our data indicate that GⅡ.14 has relatively broad binding characteristics to saliva. The potential glycanbinding mechanism of GⅡ.14 was illustrated through sequence alignment and structural comparison. Our results provide more evidence for the epidemic monitoring of NoVs of different genotypes.
[1] Ahmed S M, Hall A J, Robinson A E, et al. Global prevalence of norovirus in cases of gastroenteritis:a systematic review and meta-analysis[J]. Lancet Infect Dis, 2014, 14(8):725-730. DOI:10. 1016/S1473-3099(14)70767-4.
[2] Patel M M, Hall A J, VinjéJ, et al. Noroviruses:A comprehensive review[J]. J Clin Virol, 2009, 44(1):1-8. DOI:10. 1016/j. jcv. 2008. 10. 009.
[3] Zhang M, Fu M, Hu Q. Advances in human norovirus vaccine research[J]. Vaccines, 2021, 9(7):732. DOI:10. 3390/vaccines9070732.
[4] Kirk M D, Pires S M, Black R E, et al. World health organization estimates of the global and regional disease burden of 22 foodborne bacterial, protozoal, and viral diseases, 2010:a data synthesis[J/OL]. PLoS Medicine, 2015, 12(12):e1001921. DOI:10. 1371/journal. pmed. 1001921.
[5] Pires S M, Fischer-Walker C L, Lanata C F, et al.Aetiology-specific estimates of the global and regional incidence and mortality of diarrhoeal diseases commonly transmitted through food[J/OL]. PLoS One, 2015, 10(12):e0142927. DOI:10. 1371/journal.pone. 0142927.
[6] Lopman B A, Ozawa S, Hall A J, et al. Global economic burden of norovirus gastroenteritis[J/OL].PLoS One, 2016, 11(4):e0151219. DOI:10. 1371/journal. pone. 0151219.
[7] Vongpunsawad S, Venkataram Prasad B V, Estes M K. Norwalk virus minor capsid protein vp2 associates within the vp1 shell domain[J]. J Virol, 2013, 87(9):4818-4825. DOI:10. 1128/JVI. 03508-12.
[8] Chhabra P, De Graaf M, Parra G I, et al. Updated classification of norovirus genogroups and genotypes[J].Journal of General Virology, 2019, 100(10):1393-1406. DOI:10. 1099/jgv. 0. 001318.
[9] Kendra J A, Tohma K, Ford-Siltz L A, et al.Antigenic cartography reveals complexities of genetic determinants that lead to antigenic differences among pandemic GII. 4 noroviruses[J]. Proc Natl Acad Sci U S A, 2021, 118(11):e2015874118. DOI:10. 1073/pnas. 2015874118.
[10]Van Beek J, De Graaf M, Al-Hello H, et al. Molecular surveillance of norovirus, 2005–16:an epidemiological analysis of data collected from the NoroNet network[J].Lancet Infect Dis, 2018, 18(5):545-553. DOI:10. 1016/S1473-3099(18)30059-8.
[11]Dinu S, Nagy M, Negru D G, et al. Molecular identification of emergent GII. P17-GII. 17genotypenorovirus, Romania, 2015[J/OL]. Euro Surveill,2016,21(7):30141. DOI:10. 2807/1560-7917.ES. 2016. 21. 7. 30141.
[12]Bucardo F, Reyes Y, Becker-Dreps S, et al. Pediatric norovirus GII. 4 infections in Nicaragua, 1999–2015[J]. Infect Genet Evol, 2017, 55:305-312. DOI:10. 1016/j. meegid. 2017. 10. 001.
[13]Ao Y, Wang J, Ling H, et al. Norovirus GII. P16/GII. 2–associated gastroenteritis, China, 2016[J].Emerg Infect Dis, 2017, 23(7):1172-1175. DOI:10. 3201/eid2307. 170034.
[14]Bidalot M, Théry L, Kaplon J, et al. Emergence of new recombinant noroviruses GII. p16-GII. 4 and GII.p16-GII. 2, France, winter 2016 to 2017[J/OL]. Euro Surveill,2017,22(15):30508. DOI:10. 2807/1560-7917. ES. 2017. 22. 15. 30508.
[15]Harrington P R, Lindesmith L, Yount B, et al. Binding of norwalk virus-like particles to ABH histo-blood group antigens is blocked by antisera from infected human volunteers or experimentally vaccinated mice[J]. J Virol, 2002, 76(23):12335-12343. DOI:10. 1128/JVI. 76. 23. 12335-12343. 2002.
[16]Dabelsteen E. ABO blood group antigens in oral mucosa. What is new?[J]. J Oral Pathol Med, 2002,31(2):65-70. DOI:10. 1046/j. 0904-2512. 2001. 00004. x.
[17]Marionneau S, Cailleau-Thomas A, Rocher J, et al.ABH and Lewis histo-blood group antigens, a model for the meaning of oligosaccharide diversity in the face of a changing world[J]. Biochimie, 2001, 83(7):565-573.DOI:10. 1016/S0300-9084(01)01321-9.
[18]Singh B K, Leuthold M M, Hansman G S. Human noroviruses’ fondness for histo-blood group antigens[J]. J Virol, 2015, 89(4):2024-2040. DOI:10. 1128/JVI. 02968-14.
[19]Cao S, Lou Z, Tan M, et al. Structural basis for the recognition of blood group trisaccharides by norovirus[J]. J Virol, 2007, 81(11):5949-5957. DOI:10. 1128/JVI. 00219-07.
[20]Nooraei S, Bahrulolum H, Hoseini Z S, et al. Viruslike particles:preparation, immunogenicity and their roles as nanovaccines and drug nanocarriers[J]. J Nanobiotechnology, 2021, 19(1):59. DOI:10. 1186/s12951-021-00806-7.
[21]Jiang X, Wang M, Graham D Y, et al. Expression, self-assembly, and antigenicity of the Norwalk virus capsid protein[J]. J Virol, 1992, 66(11):6527-6532. DOI:10. 1128/jvi. 66. 11. 6527-6532. 1992.
[22]Ayyar B V, Ettayebi K, Salmen W, et al. CLIC and membrane wound repair pathways enable pandemic norovirus entry and infection[J]. Nat Commun, 2023,14(1):1148. DOI:10. 1038/s41467-023-36398-z.
[23]Zhang X-F, Huang Q, Long Y, et al. An outbreak caused by GII. 17 norovirus with a wide spectrum of HBGA-associated susceptibility[J]. Sci Rep, 2015, 5(1):17687. DOI:10. 1038/srep17687.
[24]Tan M, Jin M, Xie H, et al. Outbreak studies of a GII-3 and a GII-4 norovirus revealed an association between HBGA phenotypes and viral infection[J]. J Med Virol,2008, 80(7):1296-1301. DOI:10. 1002/jmv. 21200.
[25]Jin M, He Y, Li H, et al. Two gastroenteritis outbreaks caused by GII noroviruses:host susceptibility and HBGA phenotypes[J/OL]. PLoS One, 2013, 8(3):e58605. DOI:10. 1371/journal. pone. 0058605.
[26]宋亚贵，王万德，杨盛华，等.腹泻病毒病原电镜快速诊断及应用（附892例分析）[J].吉林医学，1998(5):13-14.
[27]丛鑫，李涵博，王鹏飞，等.诺如病毒GⅡ. 13P蛋白的糖结合特征[J].中国生物制品学杂志，2019, 32(3):261-264. DOI:10. 13200/j. cnki. cjb. 002512.
[28]Cong X, Sun X, Qi J, et al. GII. 13/21 noroviruses recognize glycans with a terminal β-galactose via an unconventional glycan binding site[J]. J Virol, 2019, 93(15):e00723-19. DOI:10. 1128/JVI. 00723-19.
[29]Chen Y, Tan M, Xia M, et al. Crystallography of a Lewis-binding norovirus, elucidation of strain-specificity to the polymorphic human histo-blood group antigens[J/OL]. PLoS Pathogens, 2011, 7(7):e1002152. DOI:10. 1371/journal. ppat. 1002152.
[30]吴铮，苏韫曦，丛鑫，等. GII. 8和GII. 9型诺如病毒P蛋白糖结合特征研究[J]. International J Virol, 2023,30(1):5-9. DOI:10. 3760/cma. j. issn. 1673-4092. 2023. 01. 002
[31]Kabue J P, Meader E, Hunter P R, et al. Genetic characterisation of Norovirus strains in outpatient children from rural communities of Vhembe district/South Africa, 2014–2015[J]. J Clin Virol, 2017, 94:100-106. DOI:10. 1016/j. jcv. 2017. 07. 005.
[32]Manso C F, Romalde J L. Molecular epidemiology of norovirus from patients with acute gastroenteritis in northwestern Spain[J]. Epidemiol Infect, 2015, 143(2):316-324. DOI:10. 1017/S0950268814000740.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004528
中图分类号:R392
引用信息:
[1]欧阳瑄泽,柴鹏弟,宋敬东等.人GⅡ.14诺如病毒VLP抗原性及组织血型抗原结合特征[J].病毒学报,2024,40(04):705-715.DOI:10.13242/j.cnki.bingduxuebao.004528.
基金信息:
北京市自然科学基金（项目号：L232011）,题目：我国诺如病毒基因变异变迁及所致胃肠炎流行特征和经济负担研究~~