致病性嗜水气单胞菌单克隆抗体的制备及特性鉴定
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摘要
嗜水气单胞菌是一种人-兽-鱼共患的条件致病菌,本研究旨在利用单克隆抗体技术,制备抗致病性嗜水气单胞菌抗体。以嗜水气单胞菌CCTCC M2014157野生型菌株免疫BALB/c小鼠,利用间接酶联免疫吸附测定技术筛选,获得能够稳定分泌抗嗜水气单胞菌单克隆抗体的杂交瘤细胞株。制备的腹水依次采用饱和硫酸铵沉淀和Protein-G柱亲和层析纯化,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析抗体纯度。制备的腹水抗体效价达16 000,纯化后(4 mg/mL)效价达8 000,亚型鉴定为IgG2a。特异性分析显示,纯化后抗体能与嗜水气单胞菌结合,与11株常见的致病菌均无交叉反应。本研究为快速检测致病性嗜水气单胞菌提供参考。
Aeromonas hydrophila is a conditional pathogen of human-beast-fish comorbidity, in this study was aiming at using monoclonal technology to prepare antipathogenic monoclonal antibody(mAb) against A. hydrophila. In this study BALB/c mice were immunized with wild type A. hydrophila strain CCTCC M2014157. A. hybridoma cell line which could stably secret monoclonal antibody(mAb) against A. hydrophila strain CCTCC M2014157, was obtained using indirect enzyme-linked immunosorbent assay(ELISA). After purification of the mAb with saturated ammonium sulfate precipitation method and protein G affinity chromatography, and the purity of mAb was analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results showed that the valence of the ascites was at 16 000, and the valence after purification(4 mg/mL) reached to 8 000, it was identified subtypically and belonged to the IgG2 a. Specificity analysis indicated the purified antibody could bind to A. hydrophila and had no cross reaction with 11 common pathogens. This study has provided a foundation for rapid detection of pathogenic A. hydrophila.
Aeromonas hydrophila is a conditional pathogen of human-beast-fish comorbidity, in this study was aiming at using monoclonal technology to prepare antipathogenic monoclonal antibody(mAb) against A. hydrophila. In this study BALB/c mice were immunized with wild type A. hydrophila strain CCTCC M2014157. A. hybridoma cell line which could stably secret monoclonal antibody(mAb) against A. hydrophila strain CCTCC M2014157, was obtained using indirect enzyme-linked immunosorbent assay(ELISA). After purification of the mAb with saturated ammonium sulfate precipitation method and protein G affinity chromatography, and the purity of mAb was analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results showed that the valence of the ascites was at 16 000, and the valence after purification(4 mg/mL) reached to 8 000, it was identified subtypically and belonged to the IgG2 a. Specificity analysis indicated the purified antibody could bind to A. hydrophila and had no cross reaction with 11 common pathogens. This study has provided a foundation for rapid detection of pathogenic A. hydrophila.
引文
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[2] 李楠,郭慧芝,焦冉,等. 草鱼的一种急性细菌性传染病病原的分离鉴定及致病性研究[J]. 水生生物学报,2011,35(6):980-987.
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[21] 翟绪昭,曾海娟,王广彬,等. 阪崎克罗诺杆菌单克隆抗体的制备及其间接竞争-ELISA检测方法[J]. 食品科学,2017,(20):268-272.
[22] 张印江,何涛,赵峰,等. 鱼类病原菌嗜水气单胞菌新菌株AhX040的分离鉴定及其拮抗菌筛选[J]. 当代水产,2017,(7):90-93.
[23] 曾海娟,王广彬,郭慧琴,等. 抗瓜类细菌性果斑病菌单克隆抗体的制备及特性鉴定[J]. 食品科学,2015,36(23):228-232.
[24] 谢曼曼,李建武,王广彬,等. 副溶血性弧菌单克隆抗体的制备及特性鉴定[J]. 食品科学,2017,38(8):63-68.
[25] Bang J,Shukla S,Kim YH,et al. Development of indirect competitive ELISA for the detection of Salmonella typhimurium[J]. Romanian Biotechnological Letters,2012,17(2):7194-7204.
[26] Pengsuk C,Chaivisuthangkura P,Longyant S,et al. Development and evaluation of a highly sensitive immunochromatographic strip test using gold nanoparticle for direct detection of Vibrio cholerae O139 in seafood samples[J]. Biosensors & Bioelectronics,2013,42(15):229-235.
[27] Charlermroj R,Himananto O,Seepiban C,et al. Multiplex detection of plant pathogens using a microsphere immunoassay technology[J]. Plos One,2013,8(4):e62344.
[28] 林鹏,卢洁,郭松林,等. 时间分辨荧光免疫分析高灵敏检测嗜水气单胞菌[J]. 化学学报,2017,75(4):398-402.
[29] Park S,Kim H,Paek SH,et al. Enzyme-linked immuno-strip biosensor to detect Escherichia coli O157?H7[J]. Ultramicroscopy,2008,108(10):1348-1351.·写作常识·
[2] 李楠,郭慧芝,焦冉,等. 草鱼的一种急性细菌性传染病病原的分离鉴定及致病性研究[J]. 水生生物学报,2011,35(6):980-987.
[3] Pridgeon JW,Klesius PH. Molecular identification and virulence of three Aeromonas hydrophila isolates cultured from infected channel catfish during a disease outbreak in west Alabama (USA) in 2009[J]. Diseases of Aquatic Organisms,2011,94(3):249-253.
[4] 洪军,白玉珍,马肖梦,等. 一株鲢鱼肠道中嗜水气单胞菌拮抗菌株的筛选、鉴定及特性[J].食品与生物技术学报,2016,35(12):1285-1291.
[5] 林金杏,杨迟,冯丽萍,等. 斑马鱼嗜水气单胞菌的鉴定和人工感染组织病理学研究[J]. 生物技术通报,2016,(9):239-245.
[6] Schnabl SM,Kunz C,Unglaub F,et al. Acute postoperative infection with Aeromonas hydrophila after using medical leeches for treatment of venous congestion[J]. Archives of Orthopaedic & Trauma Surgery,2010,130(10):1323-1328.
[7] Singh BR,Gulati BR,Virmani N,et al. Outbreak of Abortions and Infertility in Thoroughbred Mares Associated with Waterborne Aeromonas hydrophila[J]. Indian Journal of Microbiology,2011,51(2):212-216.
[8] Gold WL,Salit IE. Aeromonas hydrophila infections of skin and soft tissue: report of 11 cases and review[J]. Clinical Infectious Diseases An Official Publication of the Infectious Diseases Society of America,1993,16(1):69.
[9] Baruah FK,Ahmed NH,Grover RK. Surgical Site Infection Caused by Aeromonas hydrophila in a Patient with Underlying Malignancy[J]. Journal of Clinical & Diagnostic Research Jcdr,2015,9(1):DD01.
[10] El-Barbary MI. Some clinical, microbiological and molecular characteristics of Aeromonas hydrophila isolated from various naturally infected fishes[J]. Aquaculture International,2010,18(5):943-954.
[11] 刘训猛,习丙文,胡翔,等. 江苏地区渔源嗜水气单胞菌流行病学调查[J].安徽农业大学学报,2017,44(3):381-387.
[12] 贺文旭,毛会丽,杨利敏,等. 豫北地区主要淡水鱼类感染嗜水气单胞菌的流行病学调查[J].水产科学,2016,35(3):278-283.
[13] Ligaj M,Tichoniuk M,Gwiazdowska D,et al. Electrochemical DNA biosensor for the detection of pathogenic bacteria Aeromonas hydrophila[J]. Electrochimica Acta,2014,128(5):67-74.
[14] Hussain IA,Jeyasekaran G,Shakila RJ,et al. Detection of hemolytic strains of Aeromonas hydrophila and A. sobria along with other Aeromonas spp. from fish and fishery products by multiplex PCR[J]. Journal of Food Science & Technology,2014,51(2):401-407.
[15] 韦信贤,杨先乐,童桂香,等. 四重PCR检测致病性嗜水气单胞菌[J]. 中国人兽共患病学报,2013,29(6):587-593.
[16] 高智玲,纪雪,郭学军,等. 嗜水气单胞菌双重荧光定量PCR检测方法的建立与初步应用[J].中国人兽共患病学报,2016,32(12):1126-1130.
[17] Liu M,Mccabe E,Chapados JT,et al. Detection and identification of selected cereal rust pathogens by TaqManreal-time PCR[J]. Canadian Journal of Plant Pathology,2015,37(1):92-105.
[18] Zeng H,Guo W,Liang B,et al. Self-paired monoclonal antibody lateral flow immunoassay strip for rapid detection of Acidovorax avenae subsp. citrulli[J]. Analytical & Bioanalytical Chemistry,2016,408(22):6071-6078.
[19] Jiang D,Cao B,Wang M,et al. Development of a highly sensitive and specific monoclonal antibody based enzyme-linked immunosorbent assay for the detection of a new β-agonist, phenylethanolamine A, in food samples[J]. Journal of the Science of Food & Agriculture,2017,97(3):1001-1009.
[20] Singh S,Kumar N,Dwiwedi P,et al. Monoclonal Antibodies: A Review[J]. Current Clinical Pharmacology,2018,13(2):85-99.
[21] 翟绪昭,曾海娟,王广彬,等. 阪崎克罗诺杆菌单克隆抗体的制备及其间接竞争-ELISA检测方法[J]. 食品科学,2017,(20):268-272.
[22] 张印江,何涛,赵峰,等. 鱼类病原菌嗜水气单胞菌新菌株AhX040的分离鉴定及其拮抗菌筛选[J]. 当代水产,2017,(7):90-93.
[23] 曾海娟,王广彬,郭慧琴,等. 抗瓜类细菌性果斑病菌单克隆抗体的制备及特性鉴定[J]. 食品科学,2015,36(23):228-232.
[24] 谢曼曼,李建武,王广彬,等. 副溶血性弧菌单克隆抗体的制备及特性鉴定[J]. 食品科学,2017,38(8):63-68.
[25] Bang J,Shukla S,Kim YH,et al. Development of indirect competitive ELISA for the detection of Salmonella typhimurium[J]. Romanian Biotechnological Letters,2012,17(2):7194-7204.
[26] Pengsuk C,Chaivisuthangkura P,Longyant S,et al. Development and evaluation of a highly sensitive immunochromatographic strip test using gold nanoparticle for direct detection of Vibrio cholerae O139 in seafood samples[J]. Biosensors & Bioelectronics,2013,42(15):229-235.
[27] Charlermroj R,Himananto O,Seepiban C,et al. Multiplex detection of plant pathogens using a microsphere immunoassay technology[J]. Plos One,2013,8(4):e62344.
[28] 林鹏,卢洁,郭松林,等. 时间分辨荧光免疫分析高灵敏检测嗜水气单胞菌[J]. 化学学报,2017,75(4):398-402.
[29] Park S,Kim H,Paek SH,et al. Enzyme-linked immuno-strip biosensor to detect Escherichia coli O157?H7[J]. Ultramicroscopy,2008,108(10):1348-1351.·写作常识·