牛坏死杆菌43K OMP多克隆抗体的制备
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摘要
为了评价牛坏死杆菌外膜蛋白43K OMP的免疫原性,试验利用大肠杆菌原核表达系统对牛坏死杆菌43K OMP基因进行表达,纯化目的蛋白免疫兔,制备牛坏死杆菌43K OMP多克隆抗体,并对其进行鉴定。对基因序列进行预测分析后,针对大肠杆菌稀有密码子优化合成该基因序列,克隆至pET-32a原核表达载体上,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达及镍柱进行纯化,获得重组蛋白大小约为59 ku。重组蛋白经Western blotting鉴定后免疫兔,制备多克隆抗体。Western blot检测牛坏死杆菌43K OMP多克隆抗体与43K OMP重组蛋白具有很好的免疫原性。研究成功制备了牛坏死杆菌43K OMP多克隆抗体,为以43K OMP蛋白为基础的疫苗研究奠定基础。
In order to analyze the immunogenicity of bovine necrosis bacillus outer membrane protein(43 K OMP),the 43 K OMP gene of Fusobacterium necrophorum was expressed in E.coli prokaryotic expression system,and the purified protein was used to immunize the rabbits,polyclonal antibodies against 43 K OMP was prepared and identified.The recombinant protein was synthesized and cloned into the prokaryotic expression vector of p ET-32 a.The recombinant protein was induced by isopropylthioside β-D galactoside(IPTG)and purified by nickel column.The length of the recombinant protein was about 59 ku. Polyclonal antibody was prepared by immunization of recombinant protein with western blot.Western blot detection results showed that the 43 K OMP polyclonal antibody and 43 K OMP recombinant protein had better immunogenicity.The polyclonal antibody against bovine 43 K OMP laid a foundation for the further research of 43 K OMP protein vaccine.
In order to analyze the immunogenicity of bovine necrosis bacillus outer membrane protein(43 K OMP),the 43 K OMP gene of Fusobacterium necrophorum was expressed in E.coli prokaryotic expression system,and the purified protein was used to immunize the rabbits,polyclonal antibodies against 43 K OMP was prepared and identified.The recombinant protein was synthesized and cloned into the prokaryotic expression vector of p ET-32 a.The recombinant protein was induced by isopropylthioside β-D galactoside(IPTG)and purified by nickel column.The length of the recombinant protein was about 59 ku. Polyclonal antibody was prepared by immunization of recombinant protein with western blot.Western blot detection results showed that the 43 K OMP polyclonal antibody and 43 K OMP recombinant protein had better immunogenicity.The polyclonal antibody against bovine 43 K OMP laid a foundation for the further research of 43 K OMP protein vaccine.
引文
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[17]郭东华,王君伟,孙玉国,等.牛腐蹄病坏死梭杆菌菌株白细胞毒素基因的原核表达及其免疫活性分析[J].中国预防兽医学报,2007,29(6):435-438.
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[19]郭东华,王君伟,孙玉国,等.牛腐蹄病坏死梭杆菌白细胞毒素重组亚单位疫苗诱导小鼠免疫保护效果的观察[J].中国预防兽医学报,2008,30(5):398-402.
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[21] Li C,Ye Z,Wen L,et al.Identification of a novel vaccine candidate by immunogenic screening of Vibrio parahaemolyticus outer membrane proteins[J].Vaccine,2014,32(46):6115-6121.
[22] Liu G F,Wang J Y,Xu L W,et al. Sensitive and rapid detection of Vibrio corallilyticus by loop-mediated isothermal amplification targeted to the alpha subunit gene of RNA polymerase[J].Lett Appl Microbiol,2010,51(3):301-307.
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[24] Menon S,Pillai D K,Narayanan S.Characterization of Fusobacterium necrophorum subsp.necrophorum outer membrane proteins[J].Anaerobe,2018(1):1-2.
[25]郭明飞,关伟,李向波.抗MCM5蛋白多克隆抗体的制备及纯化[J].赤峰学院学报:自然科学版,2008(2):15.
[26] Curtis M A,Slaney J M,Carman R J,et al.Identification of the major surface protein antigens of Porphyromonas gingivalis using IgG antibody reactivity of periodontal case-control serum[J].Oral Microbiol Immunol,1991(6):321-327.
[27]李海峰,耿爽,金鑫,等.延边黄牛SREBP1基因多态性与肌内脂肪酸组成的相关性研究[J].延边大学农学学报,2017(4):68-72.
[28]徐晶,陈立志,李淑艳,等.坏死梭杆菌44.5 ku外膜蛋白的免疫原性分析[J].中国兽医科学,2016(4):512-515.
[29]王志慧.牛坏死杆菌43 k Da OMP对三种宿主细胞黏附作用的初步验证[D].大庆:黑龙江八一农垦大学,2018.
[30]王志慧,张思瑶,蒋剑成,等.牛坏死杆菌43K OMP原核表达及多克隆抗体制备[C]//中国兽医病理学2017年学术研讨会,2017.
[31]兽医病理学分会第九次全国会员代表大会论文集[M].锦州,2017:325-328.
[32]王恩雨,高晶,郭东华,等.猪整合素αv多克隆抗体制备与应用[J].黑龙江八一农垦大学学报,2017,29(4):41-44.
[2] Machado VS,Bicalho ML,Bicalho RC.Subcutaneous immunization with inactivated bacterial components and purified protein of Escherichia coli,Fusobacterium necrophorum and Trueperella pyogenes prevents puerperal metritis in Holstein dairy cows[J].PLoS One,2014,9(3):91734.
[3] Bicalho ML,Machado VS,Oikonomou G,et al.Association between virulence factors of Escherichia coli,Fusobacterium necrophorum,and Arcanobacterium pyogenes and uter ine diseases of dairy cows[J].Veterinary Microbiology,2012,157(1-2):125-131.
[4] Oikonomou G,Machado VS,Santisteban C,et al. Microbial diversity of bovine mastitic milk as described by pyrosequencing of metagenomic 16s rDNA[J].PLoS One,2012(10):47671.
[5] Qiao S,Luo Q,Zhao Y,et al. Structural basis for lipopolysaccharide insertion in the bacterial outer membrane[J].Nature,2014,511(7507):108-111.
[6] Li C,Ye Z,Wen L,et al.Identification of a novel vaccine candidate by immunogenic screening of Vibrio parahaemolyticus outer membrane proteins[J].Vaccine,2014,32(46):6115-6121.
[7] Sun D,Zhang H,Lv S,et al.Identification of a 43-kDa outer membrane protein of Fusobacterium necrophorum that exhibits similarity with pore-forming proteins of other Fusobacterium species[J].Journal of Medical Microbiology,2013,95(1):27-33.
[8]吕思文,张红,孙东波,等.牛坏死杆菌43Ku外膜蛋白截短片段的原核表达[J].中国畜牧兽医,2014,41(4):56-60.
[9] Kumar A,Peterson G,Nagaraja TG,et al.Outer membrane proteins of Fusobacterium necrophorum subsp.necrophorum and subsp.Funduliforme[J].Journal of Basic Microbiology,2014,54(8):812-817.
[10] Tadepalli S,Narayanan S G,Chengappa M,et al.Fusobacterium necrophorum:A ruminal bacterium that invades liver to cause abscesses in cattle[J].Anaerobe,2009,15(1):36-43.
[11] Paul S P,Beri R,Linney M J.Lemierre's syndrome:a sinister sore throat every clinician should remember[J].Turkish Journal of Pediatrics,2012,54(5):528-531.
[12] Zhou H T,Bennett G,Hickford J G H.Variation in Fusobacterium necrophorum strains present on the hooves of footrot infected sheep,goats and cattle[J].Veterinary Microbiology,2009,135(3-4):363-367.
[13] Narayanan SK,Nagaraja TG,Chengappa MM.Leukotoxins of gram-negative bacteria[J].Veterinary Microbiology,2002,84:337-356.
[14] Narayanan SK,Stewart GC,Chengappa MM.Fusobacterium necrophorum leukotoxin induces activation and apoptosis of bovine leukocytes[J].Infection and Immunity,2002,70(8):4609-4620.
[15] Narayanan SK,Nagaraja T G,Chengappa M M,et al.Cloning,sequencing,and expression of the leukotoxin gene from Fusobacterium necrophorum[J].Infection and Immunity,2001,69(9):5447-5455.
[16] Narayanan S K,Chengappa M M,Stewart G C,et al. Immunogenicity and protective effects of truncated recombinant leukotoxin proteins of Fusobacterium necrophorum in mice[J].Veterinary Microbiology,2003,93(4):335-347.
[17]郭东华,王君伟,孙玉国,等.牛腐蹄病坏死梭杆菌菌株白细胞毒素基因的原核表达及其免疫活性分析[J].中国预防兽医学报,2007,29(6):435-438.
[18] Guo DH,Sun DB,Wu R,et al.An indirect ELISA for serodiagnosis of cattle footrot caused by Fusobacterium necrophorum[J].Anaerobe,2010,16(4):317-320.
[19]郭东华,王君伟,孙玉国,等.牛腐蹄病坏死梭杆菌白细胞毒素重组亚单位疫苗诱导小鼠免疫保护效果的观察[J].中国预防兽医学报,2008,30(5):398-402.
[20] Qiao S,Luo Q,Zhao Y,et al.Structural basis for lipopolysaccharide insertion in the bacterial outer membrane[J].Nature,2014,7507:108-111.
[21] Li C,Ye Z,Wen L,et al.Identification of a novel vaccine candidate by immunogenic screening of Vibrio parahaemolyticus outer membrane proteins[J].Vaccine,2014,32(46):6115-6121.
[22] Liu G F,Wang J Y,Xu L W,et al. Sensitive and rapid detection of Vibrio corallilyticus by loop-mediated isothermal amplification targeted to the alpha subunit gene of RNA polymerase[J].Lett Appl Microbiol,2010,51(3):301-307.
[23] Kumar A,Menon S,Nagaraja TG,et al.Identification of an outer membrane protein of Fusobacterium necrophorum subsp.necrophorum that binds with high affinity to bovine endothelial cells[J].Veterinary Microbiology,2015,176(1-2):196-201.
[24] Menon S,Pillai D K,Narayanan S.Characterization of Fusobacterium necrophorum subsp.necrophorum outer membrane proteins[J].Anaerobe,2018(1):1-2.
[25]郭明飞,关伟,李向波.抗MCM5蛋白多克隆抗体的制备及纯化[J].赤峰学院学报:自然科学版,2008(2):15.
[26] Curtis M A,Slaney J M,Carman R J,et al.Identification of the major surface protein antigens of Porphyromonas gingivalis using IgG antibody reactivity of periodontal case-control serum[J].Oral Microbiol Immunol,1991(6):321-327.
[27]李海峰,耿爽,金鑫,等.延边黄牛SREBP1基因多态性与肌内脂肪酸组成的相关性研究[J].延边大学农学学报,2017(4):68-72.
[28]徐晶,陈立志,李淑艳,等.坏死梭杆菌44.5 ku外膜蛋白的免疫原性分析[J].中国兽医科学,2016(4):512-515.
[29]王志慧.牛坏死杆菌43 k Da OMP对三种宿主细胞黏附作用的初步验证[D].大庆:黑龙江八一农垦大学,2018.
[30]王志慧,张思瑶,蒋剑成,等.牛坏死杆菌43K OMP原核表达及多克隆抗体制备[C]//中国兽医病理学2017年学术研讨会,2017.
[31]兽医病理学分会第九次全国会员代表大会论文集[M].锦州,2017:325-328.
[32]王恩雨,高晶,郭东华,等.猪整合素αv多克隆抗体制备与应用[J].黑龙江八一农垦大学学报,2017,29(4):41-44.