单壁碳纳米管载锦鲤疱疹病毒ORF149核酸疫苗的构建

doi:10.13560/j.cnki.biotech.bull.1985.2019-0688

生物技术通报 ›› 2020, Vol. 36 ›› Issue (2): 206-213.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0688

单壁碳纳米管载锦鲤疱疹病毒ORF149核酸疫苗的构建

胡峰^1,2, 王庆¹, 李莹莹¹, 曾伟伟¹, 王高学³, 朱斌³, 王英英¹, 尹纪元¹

1. 中国水产科学研究院珠江水产研究所农业部渔药创制重点实验室广东水产动物免疫技术重点实验室,广州 510380;
2. 上海海洋大学水产与生命学院,上海 201306;
3. 西北农林科技大学动物科技学院,杨凌 712100

收稿日期:2019-07-31 出版日期:2020-02-26 发布日期:2020-02-23
作者简介:胡峰,男,硕士研究生,研究方向：鱼类病害学;E-mail：donkeyhf@126.com
基金资助:
广东省自然科学基金项目（2018A0303130029）,中国水产科学研究院珠江水产研究所基本科研业务费（2019ZX-002）,现代农业产业技术体系建设专项资金（CARS-45）

Construction of Single-walled Carbon Nanotube-loaded Koi Herpes Virus ORF149 Nucleic Acid Vaccine

HU Feng^1,2, WANG Qing¹, LI Ying-ying¹, ZENG Wei-wei¹, WANG Gao-xue³, ZHU Bin³, WANG Ying-ying¹, YIN Ji-yuan¹

1. Key Laboratory of Fishery Drug Development of Ministry of Agriculture,Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province,Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380;
2. College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306;
3.College of Animal Science and Technology,Northwest A&F University,Yangling 712100

Received:2019-07-31 Published:2020-02-26 Online:2020-02-23

摘要/Abstract

摘要： 为了开发新型高效疫苗预防锦鲤疱疹病毒病,以单壁碳纳米管（SWCNT）作为运输载体,构建锦鲤疱疹病毒（KHV）ORF149核酸疫苗。首先构建pcDNA3.1（+）-ORF149重组质粒,通过瞬时转染和免疫印迹分析确定其表达情况,然后通过1,3-偶极环加成反应法将重组质粒与SWCNTs进行偶联,最后通过扫描电镜观察和核酸电泳鉴定其偶联是否成功。结果表明,构建的重组质粒转染细胞后经免疫印迹分析和间接免疫荧光试验均能检测到特异性信号;在核酸琼脂糖凝胶电泳中,构建的重组疫苗电泳条带消失;在场发射扫描电镜观察下,与重组质粒进行偶联的SWCNTs和空白SWCNTs形态差异明显。

关键词: 单壁碳纳米管, KHV ORF149, DNA疫苗

Abstract: In order to develop a new type of highly effective vaccine against Koi Herpesvirus（KHV）disease,a single-walled carbon nanotube（SWCNT）was used as a transport vector to construct a KHV ORF149 nucleic acid vaccine. The recombinant plasmid pcDNA3.1（+）-ORF149 was firstly constructed,and its expression was confirmed by transient transfection and immunoblot analysis. Then,the recombinant plasmid was conjugated to SWCNTs by 1,3-dipolar cycloaddition reaction. Scanning electron microscopy and nucleic acid electrophoresis was conducted to identify whether the conjugation was successful. The results showed that specific signals were detected with constructed recombinant plasmid in both immunoblot analysis and indirect immunofluorescence assay after transfection. In the nucleic acid agarose gel electrophoresis,the electrophoretic band of the constructed recombinant vaccine disappeared. Under the observation of scanning electron microscopy,the morphologies of the conjugated SWCNTs with the recombinant plasmid and blank SWCNTs were significantly different.

Key words: single-walled carbon nanotubes, KHV ORF149, DNA vaccine

胡峰, 王庆, 李莹莹, 曾伟伟, 王高学, 朱斌, 王英英, 尹纪元. 单壁碳纳米管载锦鲤疱疹病毒ORF149核酸疫苗的构建[J]. 生物技术通报, 2020, 36(2): 206-213.

HU Feng, WANG Qing, LI Ying-ying, ZENG Wei-wei, WANG Gao-xue, ZHU Bin, WANG Ying-ying, YIN Ji-yuan. Construction of Single-walled Carbon Nanotube-loaded Koi Herpes Virus ORF149 Nucleic Acid Vaccine[J]. Biotechnology Bulletin, 2020, 36(2): 206-213.

参考文献

[1] Hedrick RP, Gilad O, Yun S, et al.A herpesvirus associated with mass mortality of juvenile and adult koi, a strain of common carp[J]. Journal of Aquatic Animal Health, 2000, 12(1):44-57.
[2] Gudding R, Van Muiswinkel WB.A history of fish vaccination:science-based disease prevention in aquaculture[J]. Fish Shellfish Immunol, 2013, 35(6):1683-1689.
[3] Sommerset I, Krossoy B, Biering E, et al.Vaccines for fish in aquaculture[J]. Expert Rev Vaccines, 2005, 4(1):89-101.
[4] Hastein T, Gudding R, Evensen O.Bacterial vaccines for fish-An update of the current situation worldwide[J]. Fish Vaccinology, 2005, 25(3):55-74.
[5] 张子鹏, 余涛. 锦鲤疱疹病毒ORF30和ORF131蛋白的原核表达及纯化[J]. 中国农业信息, 2015, 10(5):135-142.
[6] Evensen O, Leong JA.DNA vaccines against viral diseases of farmed fish[J]. Fish Shellfish Immunol, 2013, 35(6):1751-1763.
[7] Anderson ED, Mourich DV, Fahrenkrug SC, et al.Genetic immunization of rainbow trout(Oncorhynchus mykiss)against infectious hematopoietic necrosis virus[J]. Mol Mar Biol Biotechnol, 1996, 5(2):114-122.
[8] Lorenzen N, Lorenzen E, Einer-Jensen K, et al.Protective immunity to VHS in rainbow trout(Oncorhynchus mykiss, Walbaum)following DNA vaccination[J]. Fish Shellfish Immunol, 1998, 8(4):261-270.
[9] Sommerset I, Skern R, Biering E, et al.Protection against Atlantic halibut nodavirus in turbot is induced by recombinant capsid protein vaccination but not following DNA vaccination[J]. Fish Shellfish Immunol, 2005, 18(1):13-29.
[10] Mikalsen AB, Torgersen J, Alestrom P, et al.Protection of Atlantic salmon Salmo salar against infectious pancreatic necrosis after DNA vaccination[J]. Diseases of Aquatic Organisms, 2004, 60(1):11-20.
[11] Lorenzen E, Einer-Jensen K, Rasmussen JS, et al.The protective mechanisms induced by a fish rhabdovirus DNA vaccine depend on temperature[J]. Vaccine, 2009, 27(29):3870-3880.
[12] Emmenegger EJ, Kurath G.DNA vaccine protects ornamental koi(Cyprinus carpio koi)against north American spring viremia of carp virus[J]. Vaccine, 2008, 26(50):6415-6421.
[13] Zhou JX, Wang H, Li XW, et al.Construction of KHV-CJ ORF25 DNA vaccine and immune challenge test[J]. Journal of Fish Diseases, 2014, 37(4):319-325.
[14] Zhou J, Xue J, Wang Q, et al.Vaccination of plasmid DNA encoding ORF81 gene of CJ strains of KHV provides protection to immunized carp in vitro[J]. Cell Dev Biol Anim, 2014, 50(6):489-495.
[15] Davison AJ, Eberle R, Ehlers B, et al.The order herpesvirales[J]. Arch Virol, 2009, 154(1):171-175.
[16] Aoki T, Hirono I, Kurokawa K, et al.Genome sequences of three koi herpesvirus isolates representing the expanding distribution of an emerging disease threatening koi and common carp worldwide[J]. Journal of Virology, 2007, 81(10):5058-5065.
[17] Zhang Q, Gui JF.Virus genomes and virus-host interactions in aquaculture animals[J]. Science China Life Sciences, 2015, 58(2):156-169.
[18] Zeng XT, Chen ZY, Deng YS, et al.Complete genome sequence and architecture of crucian carp Carassius auratus herpesvirus(CaHV)[J]. Archives of Virology, 2016, 161(12):3577-3581.
[19] Yi Y, Zhang H, Lee X, et al.Extracellular virion proteins of two Chinese CyHV-3/KHV isolates, and identification of two novel envelope proteins[J]. Virus Research, 2014, 191(5):108-116.
[20] Michel B, Leroy B, Stalin Raj V, et al.The genome of cyprinid herpesvirus 3 encodes 40 proteins incorporated in mature virions[J]. J Gen Virol, 2010, 91(2):452-462.
[21] Micheal S, Hogewind BF, Khan MI, et al.Variants in the PRPF8 gene are associated with glaucoma[J]. Mol Neurobiol, 2018, 55(5):4504-4510.
[22] Kattlun J, Menanteau-Ledouble S, Gotesman M, et al.Immunogenic potential of a membrane protein encoded by the viral gene located at ORF 81 of Cyprinid Herpes-virus-3[J]. Wien Tierarz Monats, 2016, 103(3-4):57-67.
[23] Fuchs W, Granzow H, Dauber M, et al.Identification of structural proteins of koi herpesvirus[J]. Arch Virol, 2014, 159(12):3257-3268.
[24] Torrent F, Villena A, Lee PA, et al.The amino-terminal domain of ORF149 of koi herpesvirus is preferentially targeted by IgM from carp populations surviving infection[J]. Arch Virol, 2016, 161(10):2653-2665.
[25] Monaghan SJ, Thompson KD, Bron JE, et al.Expression of immunogenic structural proteins of cyprinid herpesvirus 3 in vitro assessed using immunofluorescence[J]. Vet Res, 2016, 47:8.
[26] Adomako M, St-Hilaire S, Zheng Y, et al.Oral DNA vaccination of rainbow trout, Oncorhynchus mykiss(Walbaum), against infectious haematopoietic necrosis virus using PLGA[Poly(D, L-Lactic-Co-Glycolic Acid)]nanoparticles[J]. J Fish Dis, 2012, 35(3):203-214.
[27] Rivas-Aravena A, Fuentes Y, Cartagena J, et al.Development of a nanoparticle-based oral vaccine for Atlantic salmon against ISAV using an alphavirus replicon as adjuvant[J]. Fish Shellfish Immunol, 2015, 45(1):157-166.
[28] Tang N, Zhong W, Au C, et al.Large-scale synthesis, annealing, purification, and magnetic properties of crystalline helical carbon nanotubes with symmetrical structures[J]. Advanced Functional Materials, 2007, 17(9):1542-1550.
[29] Deng X, Wu F, Liu Z, et al.The splenic toxicity of water soluble multi-walled carbon nanotubes in mice[J]. Carbon, 2009, 47(6):1421-1428.
[30] Georgakilas V, Tagmatarchis N, Pantarotto D, et al.Amino acid functionalisation of water soluble carbon nanotubes[J]. Chem Commun(Camb), 2002, 56(24):3050-3051.
[31] Pantarotto D, Singh R, McCarthy D, et al. Functionalized carbon nanotubes for plasmid DNA gene delivery[J]. Angew Chem Int Ed Engl, 2004, 43(39):5242-5246.
[32] Perelberg A, Ronen A, Hutoran M, et al.Protection of cultured Cyprinus carpio against a lethal viral disease by an attenuated virus vaccine[J]. Vaccine, 2005, 23(26):3396-3403.
[33] Foldvari M, Bagonluri M.Carbon nanotubes as functional excipients for nanomedicines:II. Drug delivery and biocompatibility issues[J]. Nanomedicine, 2008, 4(2):183-200.
[34] Scheinberg DA, Mr M, Dao T, et al.Carbon nanotubes as vaccine scaffolds[J]. Adv Drug Deliv Rev, 2013, 65(13):2016-2022.
[35] Yandar N, Pastorin G, Prato M, et al.Immunological profile of a Plasmodium vivax AMA-1 N-terminus peptide-carbon nanotube conjugate in an infected Plasmodium berghei mouse model[J]. Vaccine, 2008, 26(46):5864-5873.
[36] Parra J, Abad-Somovilla A, Mercader JV, et al.Carbon nanotube-protein carriers enhance size-dependent self-adjuvant antibody response to haptens[J]. Journal of Controlled Release, 2013, 170(2):242-251.
[37] Villa CH, Dao T, Ahearn I, et al.Single-walled carbon nanotubes deliver peptide antigen into dendritic cells and enhance IgG responses to tumor-associated antigens[J]. ACS Nano, 2011, 5(7):5300-5311.
[38] Wang Y, Liu GL, Li DL, et al.The protective immunity against grass carp reovirus in grass carp induced by a DNA vaccination using single-walled carbon nanotubes as delivery vehicles[J]. Fish Shellfish Immunol, 2015, 47(2):732-742.
[39] Gong YX, Zhu B, Liu GL, et al.Single-walled carbon nanotubes as delivery vehicles enhance the immunoprotective effects of a recombinant vaccine against Aeromonas hydrophila[J]. Fish Shellfish Immunol, 2015, 42(1):213-220.
[40] Zhang C, Li LH, Wang J, et al.Enhanced protective immunity against spring viremia of carp virus infection can be induced by recombinant subunit vaccine conjugated to single-walled carbon nanotubes[J]. Vaccine, 2018, 36(42):6334-6344.
[41] Zhang C, Zhao Z, Liu GY, et al.Immune response and protective effect against spring viremia of carp virus induced by intramuscular vaccination with a SWCNTs-DNA vaccine encoding matrix protein[J]. Fish Shellfish Immunol, 2018, 79(12):256-264.
[42] Perelberg A, Ilouze M, Kotler M, et al.Antibody response and resistance of Cyprinus carpio immunized with cyprinid herpes virus 3(CyHV-3)[J]. Vaccine, 2008, 26(30):3750-3756.
[43] Cui LC, Guan XT, Liu ZM, Tian CY, et al.Recombinant lactobacillus expressing G protein of spring viremia of carp virus(SVCV)combined with ORF81 protein of koi herpesvirus(KHV):A promising way to induce protective immunity against SVCV and KHV infection in cyprinid fish via oral vaccination[J]. Vaccine, 2015, 33(27):3092-3099.
[44] Embregts CWE, Tadmor-Levi R, Veselý T, Pokorová D, et al.Intra-muscular and oral vaccination using a Koi Herpesvirus ORF25 DNA vaccine does not confer protection in common carp(Cyprinus carpio L.)[J]. Fish Shellfish Immunol, 2019, 85:90-98.

单壁碳纳米管载锦鲤疱疹病毒ORF149核酸疫苗的构建

Construction of Single-walled Carbon Nanotube-loaded Koi Herpes Virus ORF149 Nucleic Acid Vaccine

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