2024 05 v.40 970-977
猴痘病毒A29L-A35R-M1R融合蛋白的原核表达、纯化及免疫原性初步分析
基金项目(Foundation):
国家重点研发计划（项目号：2023YFD1800404）,题目：猴痘候选疫苗、抗体研制~~
邮箱(Email):
lnjzmmx@163.com;dlfei7712@126.com;
DOI:
10.13242/j.cnki.bingduxuebao.004573
中文作者单位:
锦州医科大学畜牧兽医学院;辽宁省农业发展服务中心;
摘要(Abstract):
猴痘病毒（Monkeypox virus,MPXV）是一种能够感染人和动物的痘病毒，已对人类公共卫生安全构成巨大威胁，安全高效的疫苗能够有效预防和控制病毒的传播。本研究选取MPXV A29L、A35R和M1R氨基酸序列通过柔性Linker(GGGGS)连接设计融合蛋白，并根据大肠杆菌密码子偏嗜性对其密码子优化后合成全基因，插入pET-28a载体，构建pET28a-A29L-A35R-M1R重组质粒。转化至E.coli BL21(DE3)感受态细胞中诱导表达，经SDSPAGE和Western blot进行鉴定，并确定最佳诱导条件。在此基础上，对融合蛋白rA29L-A35R-M1R进行Ni-NTA亲和层析柱纯化后，免疫小鼠。通过ELISA方法检测抗MPXV A29L、A35R和M1R特异性IgG和细胞因子（IFN-γ、IL-2和IL-4）水平，评价融合蛋白免疫原性。实验结果显示：重组质粒pET-28a-A29L-A35R-M1R在E.coli BL21(DE3)感受态细胞中成功表达，最佳表达条件为37℃、0.25 mmol/L IPTG诱导5 h；将纯化的融合蛋白rA29LA35R-M1R免疫小鼠3次后，能够产生抗A29L、A35R和M1R特异性IgG，其中anti-rA29L-A35R-M1R IgG水平最高，且融合蛋白rA29L-A35R-M1R能够刺激机体产生高水平IFN-γ,IL-2和IL-4，与对照组差异均具有统计学意义（P<0.01）。由此表明，融合蛋白rA29L-A35R-M1R具有良好的免疫原性。为猴痘病毒新型疫苗和治疗性生物制剂的研发提供理论依据。
关键词(KeyWords):
猴痘病毒;;融合蛋白;;原核表达;;免疫原性
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参考文献
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[24]Huang Z, Li G, Zhang C, et al. A study on the effects of linker flexibility on acid phosphatase PhoC-GFP fusion protein using a novel linker library[J]. Enzyme Microb Technol, 2016, 83:1-6. DOI:10. 1016/j.enzmictec. 2015. 11. 002.
[2]吕诗韵，卢佳，李新国，等.猴痘病毒及其疫苗的研究进展[J].中国生物制品学杂志，2022, 35(12):1409-1413.
[3] Fogg C, Lustig S, Whitbeck JC, et al. Protective immunity to vaccinia virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions[J]. J Virol, 2004,78(19):10230-10237. DOI:10. 1128/jvi. 78. 19. 10230-10237. 2004.
[4] Hooper JW, Thompson E, Wilhelmsen C, et al.Smallpox DNA vaccine protects nonhuman Primates against lethal monkeypox[J]. J Virol, 2004, 78(9):4433-4443. DOI:10. 1128/jvi. 78. 9. 4433-4443. 2004.
[5] Hou F, Zhang Y, Liu X, et al. mRNA vaccines encoding fusion proteins of monkeypox virus antigens protect mice from vaccinia virus challenge[J]. Nat Commun, 2023, 14:5925. DOI:10. 1038/s41467-023-41628-5.
[6] Likos AM, Sammons SA, Olson VA, et al. A tale of two clades:monkeypox viruses[J]. J Gen Virol, 2005,86(pt 10):2661-2672. DOI:10. 1099/vir. 0. 81215-0.
[7] Ramírez JC, Tapia E, Esteban M. Administration to mice of a monoclonal antibody that neutralizes the intracellular mature virus form of vaccinia virus limits virus replication efficiently under prophylactic and therapeutic conditions[J]. J Gen Virol, 2002, 83(pt5):1059-1067. DOI:10. 1099/0022-1317-83-5-1059.
[8] Li M, Ren Z, Wang Y, et al. Three neutralizing mAbs induced by MPXV A29L protein recognizing different epitopes act synergistically against orthopoxvirus[J].Emerg Microbes Infect, 2023, 12(2):2223669. DOI:10. 1080/22221751. 2023. 2223669.
[9] Priyamvada L, Satheshkumar PS. Variola and monkeypox viruses(Poxviridae)[M]//Encyclopedia of Virology. Amsterdam:Elsevier, 2021:868-874. DOI:10. 1016/b978-0-12-809633-8. 21545-8.
[10]石建州，王国川，王彦伟，等.猴痘病毒研究进展[J].病毒学报，2022, 38(5):1195-1205. DOI:10. 13242/j. cnki. bingduxuebao. 004188.
[11]Adler H, Gould S, Hine P, et al. Clinical features and management of human monkeypox:a retrospective observational study in the UK[J]. Lancet Infect Dis,2022, 22(8):1153-1162. DOI:10. 1016/S1473-3099(22)00228-6.
[12]McFadden G. Poxvirus tropism[J]. Nat Rev Microbiol, 2005, 3(3):201-213. DOI:10. 1038/nrmicro1099.
[13]Ogoina D, Izibewule JH, Ogunleye A, et al. The 2017human monkeypox outbreak in Nigeria-Report of outbreak experience and response in the Niger Delta University Teaching Hospital, Bayelsa State, Nigeria[J]. PLoS One, 2019, 14(4):e0214229. DOI:10. 1371/journal. pone. 0214229.
[14]Hatmal MM, Al-Hatamleh MAI, Olaimat AN, et al.Comprehensive literature review of monkeypox[J].Emerg Microbes Infect, 2022, 11(1):2600-2631.DOI:10. 1080/22221751. 2022. 2132882.
[15]Bunge EM, Hoet B, Chen L, et al. The changing epidemiology of human monkeypox-a potential threat?A systematic review[J]. PLoS Negl Trop Dis, 2022,16(2):e0010141. DOI:10. 1371/journal.pntd. 0010141.
[16]Happi C, Adetifa I, Mbala P, et al. Urgent need for a non-discriminatory and non-stigmatizing nomenclature for monkeypox virus[J]. PLoS Biol, 2022, 20(8):e3001769. DOI:10. 1371/journal. pbio. 3001769.
[17]祁贤.猴痘病毒进化及检测技术研究进展[J].江苏预防医学，2023, 34(1):1-7. DOI:10. 13668/j.issn. 1006-9070. 2023. 01. 001.
[18]Isidro J, Borges V, Pinto M, et al. Phylogenomic characterization and signs of microevolution in the 2022multi-country outbreak of monkeypox virus[J]. Nat Med, 2022, 28:1569-1572. DOI:10. 1038/s41591-022-01907-y.
[19]王雪琛，李涛，王慧，等.猴痘病毒的研究进展概述[J].病毒学报，2024, 40(1):169-182. DOI:10. 13242/j. cnki. bingduxuebao. 004440.
[20]Volkmann A, Williamson AL, Weidenthaler H, et al.The Brighton Collaboration standardized template for collection of key information for risk/benefit assessment of a Modified Vaccinia Ankara(MVA)vaccine platform[J]. Vaccine, 2021, 39(22):3067-3080. DOI:10. 1016/j. vaccine. 2020. 08. 050.
[21]Rao AK, Petersen BW, Whitehill F, et al. Use of JYNNEOS(smallpox and monkeypox vaccine, live,nonreplicating)for preexposure vaccination of persons at risk for occupational exposure to orthopoxviruses:recommendations of the advisory committee on immunization practices-United States, 2022[J].MMWR Morb Mortal Wkly Rep, 2022, 71(22):734-742. DOI:10. 15585/mmwr. mm7122e1.
[22]Brown K, Leggat PA. Human monkeypox:current state of knowledge and implications for the future[J].Trop Med Infect Dis, 2016, 1(1):E8. DOI:10. 3390/tropicalmed1010008.
[23]Ruan D, He J, Li CF, et al. Skp2 deficiency restricts the progression and stem cell features of castrationresistant prostate cancer by destabilizing Twist[J].Oncogene, 2017, 36(30):4299-4310. DOI:10. 1038/onc. 2017. 64.
[24]Huang Z, Li G, Zhang C, et al. A study on the effects of linker flexibility on acid phosphatase PhoC-GFP fusion protein using a novel linker library[J]. Enzyme Microb Technol, 2016, 83:1-6. DOI:10. 1016/j.enzmictec. 2015. 11. 002.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004573
中图分类号:R392
引用信息:
[1]张晓凤,高志峰,王紫研等.猴痘病毒A29L-A35R-M1R融合蛋白的原核表达、纯化及免疫原性初步分析[J].病毒学报,2024,40(05):970-977.DOI:10.13242/j.cnki.bingduxuebao.004573.
基金信息:
国家重点研发计划（项目号：2023YFD1800404）,题目：猴痘候选疫苗、抗体研制~~
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