大口黑鲈弹状病毒G蛋白胞外区原核表达及多克隆抗体制备

doi:10.16431/j.cnki.1671-7236.2023.09.032

摘要/Abstract

摘要： 【目的】获得能特异性识别大口黑鲈弹状病毒(Micropterus salmoides rhabdovirus,MSRV)G蛋白的多克隆抗体,为后续MSRV双抗夹心法胶体金试纸条的研制提供材料。【方法】根据GenBank上MSRV G蛋白的基因序列(GenBank登录号:OK272491.1)设计扩增其胞外区的特异性引物,以感染MSRV的GS细胞的cDNA为模板,通过PCR扩增获得目的序列,双酶切后连接至pET-28a(+)表达载体构建重组质粒pET-28a-ΔG,将重组质粒转化大肠杆菌Trans5α感受态细胞。挑取测序正确的阳性克隆提取重组表达质粒pET-28a-ΔG,转化大肠杆菌BL21(DE3)感受态细胞,再次挑取阳性克隆,测序正确后用IPTG诱导表达。目的蛋白经Ni-NTA树脂层析柱纯化后免疫BALB/c雌性小鼠,4次免疫后眼眶采血获得抗MSRV G蛋白的多克隆抗体,最后通过ELISA、Western blotting和间接免疫荧光试验(IFA)鉴定多克隆抗体的效价和特异性。【结果】PCR结果显示,获得大小为1 323 bp的目的条带。SDS-PAGE结果显示,构建的重组表达质粒可高效表达目的蛋白,Ni柱纯化后获得单一目的蛋白,分子质量在48.5 ku左右,与预期相符,且纯度符合免疫原要求。ELISA结果显示,制备的多克隆抗体效价为1∶204 800。Western blotting结果显示,制备的多克隆抗体可特异性识别不同批次的MSRV G蛋白。IFA结果显示,制备的多克隆抗体能特异性识别天然状态下的G蛋白。【结论】本研究利用MSRV G蛋白的胞外区重组蛋白成功制备出能特异性识别MSRV G蛋白的多克隆抗体,为后续MSRV的免疫检测和疫苗开发奠定了基础。

关键词: 大口黑鲈弹状病毒; G蛋白; 原核表达; 多克隆抗体

Abstract: 【Objective】 The purpose of this experiment was to obtain polyclonal antibodies that could specifically recognize the G protein of Micropterus salmoides rhabdovirus (MSRV),and provide materials for the subsequent development of the MSRV double antibody sandwich colloidal gold strip.【Method】 Based on the genome sequence of MSRV (GenBank No.:OK272491.1),specific primers were designed to amplify the extracellular region of G protein of MSRV.The G gene was amplified via PCR using the cDNA from MSRV infected GS cells,and then ligated into the pET-28a(+) expression vector after double digestion to construct pET-28a-ΔG recombinant plasmid.The recombinant plasmid was transformed into Escherichia coli Trans5α competent cells and the positive clone was identified by DNA sequencing.Then,the plasmid pET-28a-ΔG was extracted and transformed into the Escherichia coli BL21(DE3) competent cells.Positive clones were selected again,and the expression was induced by IPTG after correct sequencing. The target protein was purified by Ni-NTA resin chromatography column and used as an immunogen to immunize female BALB/c mice.Polyclonal antibodies against MSRV G protein were obtained by orbital blood collection after four immunizations.Finally,the titer and specificity of the obtained polyclonal antibodies were characterized by ELISA,Western blotting and indirect immunofluorescence assay (IFA).【Result】 PCR results showed that a target band with a size of 1 323 bp was obtained.SDS-PAGE results showed that the recombinant plasmid could efficiently express the target protein,and a single target protein was obtained after Ni column purification,with a molecular weight of about 48.5 ku,which was consistent with expectations,and the purity met the immunogenic requirements.ELISA results showed that the titer of the prepared polyclonal antibody was 1∶204 800.Western blotting results showed that the prepared polyclonal antibodies could specifically recognize different batches of MSRV G proteins.IFA results showed that the prepared polyclonal antibody could specifically recognize G protein in its natural state. 【Conclusion】 In this study,the recombinant protein of the extracellular region of MSRV G protein was successfully used to prepare polyclonal antibodies that could specifically recognize MSRV G protein,which laid a foundation for subsequent MSRV immune detection and vaccine development.

Key words: Micropterus salmoides rhabdovirus; G protein; prokaryotic expression; polyclonal antibodies

中图分类号:

S852.65^＋9.5

刘海翔, 张佩佩, 邓思, 秦英惠, 姚伦广. 大口黑鲈弹状病毒G蛋白胞外区原核表达及多克隆抗体制备[J]. 中国畜牧兽医, 2023, 50(9): 3762-3770.

LIU Haixiang, ZHANG Peipei, DENG Si, QIN Yinghui, YAO Lunguang. Prokaryotic Expression and Polyclonal Antibody Preparation of Extracellular Region of G Protein of Micropterus salmoides Rhabdovirus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3762-3770.

参考文献

[1] DIETZGEN R G,KONDO H,GOODIN M M,et al.The family Rhabdoviridae:Mono- and bipartite negative-sense RNA viruses with diverse genome organization and common evolutionary origins[J].Virus Research,2017,227:158-170.
[2] KUZMIN I V,NOVELLA I S,DIETZGEN R G,et al.The Rhabdoviruses:Biodiversity,phylogenetics,and evolution[J].Infection, Genetics and Evolution,2009,9(4):541-553.
[3] ASSENBERG R,DELMAS O,MORIN B,et al.Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription[J].Antiviral Research,2010,87(2):149-161.
[4] CHEN Z Y,GAO X C,ZHANG Q Y.Whole-genome analysis of a novel fish Reovirus (MsReV) discloses Aquareovirus genomic structure relationship with host in saline environments[J]. Viruses,2015,7(8):4282-4302.
[5] HOFFMANN B,BEER M,SCHVTZE H,et al.Fish rhabdoviruses:Molecular epidemiology and evolution[J].Current Topics in Microbiology and Immunology,2005,292:81-117.
[6] GALINIER R,VAN B S,AMILHAT E,et al.Complete genomic sequence and taxonomic position of Eel virus European X (EVEX),a Rhabdovirus of European eel[J].Virus Research,2012,166(1-2):1-12.
[7] ZHANG L J,LI N Q,LIN Q,et al.An avirulent Micropterus salmoides rhabdovirus vaccine candidate protects Chinese perch against Rhabdovirus infection[J].Fish & Shellfish Immunology,2018,77:474-480.
[8] 雷燕,戚瑞荣,崔龙波,等.大口黑鲈鱼种弹状病毒病的诊断[J].大连海洋大学学报,2015,30(3):305-308. LEI Y,QI R R,CUI L B,et al.Diagnosis of Rhabdovirus disease in juvenile largemouth bass Micropterus salmonides[J].Journal of Dalian Ocean University,2015,30(3):305-308.(in Chinese)
[9] MA D,DENG G,BAI J,et al.A strain of Siniperca chuatsi rhabdovirus causes high mortality among cultured largemouth bass in South China[J].Journal of Aquatic Animal Health,2013,25(3):197-204.
[10] FU X Z,LIN Q,LIANG H R,et al.The biological features and genetic diversity of novel Fish rhabdovirus isolates in China[J].Archives of Virology,2017,162(9):2829-2834.
[11] QIN Y H,ZHANG P P,ZHANG M F,et al.Isolation and identification of a new strain Micropterus salmoides rhabdovirus (MSRV) from largemouth bass Micropterus salmoides in China[J].Aquaculture,2023,572:739538.
[12] JAYAKAR H R,JEETENDRA E,WHITT M A.Rhabdovirus assembly and budding[J].Virus Research,2004,106(2):117-132.
[13] 楼允东.我国鱼类引种研究的现状与对策[J].水产学报,2000,24(2):185-192. LOU Y D.Present situation and countermeasure of the study on fish introduction in China[J].Journal of Fisheries of China,2000,24(2):185-192.(in Chinese)
[14] 吴锐全,肖学铮,冯启新.广东优势淡水水产品的养殖现状和竞争力分析[J].广东饲料,2006,15(2):11-14. WU R Q,XIAO X Z,FENG Q X.Analysis on the current situation and competitiveness of Guangdong’s dominant freshwater aquatic products breeding[J].Guangdong Feed,2006,15(2):11-14.(in Chinese)
[15] LYU S J,YUAN X M,ZHANG H Q,et al.Isolation and characterization of a novel strain (YH01) of Micropterus salmoides rhabdovirus and expression of its glycoprotein by the Baculovirus expression system[J].Journal of Zhejiang University,2019,20(9):728-739.
[16] YI W,ZHANG X,ZENG K,et al.Construction of a DNA vaccine and its protective effect on largemouth bass (Micropterus salmoides) challenged with Largemouth bass virus (LMBV)[J].Fish & Shellfish Immunology,2020,106:103-109.
[17] CAI J,YU D,XIA H,et al.Identification and characterization of a Nervous necrosis virus isolated from largemouth bass (Micropterus salmoides)[J]. Journal of Fish Diseases,2022,45(4):607-611.
[18] GUO Z R,ZHAO Z,ZHANG C,et al.Carbon nanotubes-loaded subunit vaccine can increase protective immunity against Rhabdovirus infections of largemouth bass (Micropterus salmoides)[J].Fish & Shellfish Immunology,2020,99:548-554.
[19] JIN Y,YANG F,ZHANG G,et al.Synthesized magnolol derivatives improve anti-Micropterus salmoides rhabdovirus (MSRV) activity in vivo[J].Viruses,2022,14(7):1421.
[20] HU Y,SHAN L,QIU T,et al.Synthesis and biological evaluation of novel coumarin derivatives in rhabdoviral clearance[J].European Journal of Medicinal Chemistry,2021,223:113739.
[21] LI G,WANG A P,CHEN Y M,et al.Development of a colloidal gold-based immunochromatographic strip for rapid detection of Severe acute respiratory syndrome coronavirus 2 spike protein[J].Frontiers in Immunology,2021,12:635677.
[22] ZHANG X Y,LIU X Y,WU X D,et al.A colloidal gold test strip assay for the detection of African swine fever virus based on two monoclonal antibodies against p30[J].Archives of Virology,2021,166(3):871-879.
[23] CHEN L L,SUN Y N,HU X F,et al.Colloidal gold-based immunochromatographic strip assay for the rapid detection of diminazene in milk[J].Food Additives & Contaminants.Part A,Chemistry,Analysis,Control,Exposure & Risk Assessment,2020,37(10):1667-1677.
[24] 蒋亚君,陈世钰,鑫婷,等.非洲猪瘟病毒360-12L蛋白的原核表达及其多克隆抗体制备[J].中国畜牧兽医,2022,49(1):352-360. JIANG Y J,CHEN S Y,XIN T,et al.Prokaryotic expression of African swine fever virus 360-12L protein and preparation of its polyclonal antibody[J].China Animal Husbandry & Veterinary Medicine,2022,49(1):352-360.(in Chinese)
[25] 刘雪婷,王召阳,鑫婷,等.非洲猪瘟病毒B438L蛋白的原核表达及其多克隆抗体的制备与鉴定[J].中国畜牧兽医,2021,48(3):991-1000. LIU X T,WANG Z Y,XIN T,et al.Prokaryotic expression and polyclonal antibody preparation and identification of African swine fever virus B438L protein[J].China Animal Husbandry & Veterinary Medicine,2021,48(3):991-1000.(in Chinese)
[26] HASSANTABAR F,ZORRIEHZAHRA M J,FIROUZBAKHSH F,et al.Development and evaluation of colloidal gold immunochromatography test strip for rapid diagnosis of Nervous necrosis virus in golden grey mullet (Chelon aurata)[J]. Journal of Fish Diseases,2021,44(6):783-791.
[27] TANG X Q,CAO J,ZHANG J L,et al.Development of monoclonal antibody against glycoprotein of Hirame novirhabdovirus (HIRRV) with virus neutralizing activity[J].Microbial Pathogenesis,2021,154:104868.
[28] ZHAO L N,TANG X Q,SHENG X Z,et al.Different immune responses of flounder (Paralichthys olivaceus) towards the full-length and N-terminal or C-terminal portion of Hirame novirhabdovirus glycoprotein[J].Fish & Shellfish Immunology,2020,104:279-288.
[29] CHEN Z Y,LEI X Y,ZHANG Q Y.The antiviral defense mechanisms in mandarin fish induced by DNA vaccination against a Rhabdovirus[J].Veterinary Microbiology,2012,157(3-4):264-275.