首页 >  水产科学 >  大口黑鲈免疫球蛋白单克隆抗体的制备及初步应用

2022, 41(4): 670-675. doi: 10.16378/j.cnki.1003-1111.20238

大口黑鲈免疫球蛋白单克隆抗体的制备及初步应用

1. 吉林农业大学 动物科学与技术学院, 吉林省动物微生态制剂工程研究中心, 吉林 长春 130118;

2. 浙江省淡水水产研究所, 农业农村部淡水渔业健康养殖重点实验室, 浙江省鱼类健康与营养重点实验室, 浙江 湖州 313001;

3. 金华市水产技术推广站, 浙江 金华, 321051

收稿日期:2020-11-04
修回日期:2021-02-05

基金项目:   金华市科技计划项目(2020-2-025).  浙江省属院所扶持专项(2020YSZX001)  湖州市公益性技术应用研究项目(2017GY08) 

关键词: 大口黑鲈 , 免疫球蛋白 , 单克隆抗体 , 抗体效价

Preparation and Application of Immunoglobulin Monoclonal Antibody from Largemouth Bass Micropterus salmoides

1. Jilin Provincial Engineering Research Center of Animal Probiotics, College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China;

2. Key Laboratory of Fish Health and Nutrition of Zhejiang Province, Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture and Rural Affairs, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China;

3. Jinhua Fisheries Technology Promotion Station, Jinhua 321051, China

Received Date:2020-11-04
Accepted Date:2021-02-05

Keywords: Micropterus salmoides , immunoglobulin , monoclonal antibody , antibody titer

利用饱和硫酸铵盐析法和亲和柱层析法提取制备大口黑鲈血清的免疫球蛋白,进而通过常规方法4次免疫BALB/c小鼠,取脾脏组织细胞与小鼠骨髓瘤Sp2/0细胞进行融合,经杂交瘤细胞筛选和单克隆化细胞培养制备单克隆抗体,并利用酶联免疫吸附法测定其抗体效价、灵敏度以及特异性反应。制备甲醛灭活大口黑鲈虹彩病毒疫苗进而免疫大口黑鲈,利用制备的单克隆抗体检测免疫后的抗体水平。制备获得2株大口黑鲈免疫球蛋白单克隆抗体杂交瘤细胞A9E7和C9B9,其腹水抗体效价可达1∶2.187×106和1∶7.29×105,两者对大口黑鲈免疫球蛋白的灵敏度为20 ng和10 ng,交叉反应试验结果表明,A9E7和C9B9均能与大口黑鲈免疫球蛋白特异性结合,A9E7与花鲈血清可发生交叉反应,而C9B9可与鳜血清发生交叉反应,且两者与鲫、草鱼、团头鲂、翘嘴鲌、黄颡鱼、光唇鱼、黄鳝、泥鳅等血清免疫球蛋白无交叉反应。免疫大口黑鲈虹彩病毒后,大口黑鲈体内抗体水平显著增加并于35 d时达到峰值,其抗体效价可达1∶1280。具有特异性强、灵敏度高的大口黑鲈免疫球蛋白单克隆抗体可用于大口黑鲈病害的早期病害监测、免疫应答规律研究和疫苗研发。

Serum immunoglobulin from largemouth bass Micropterus salmoides was purified by saturated ammonium sulfate and followed by affinity column chromatography extraction, then BALB/c mice were immunized with purified immunoglobulin, and the immunized spleen cells were fused with Sp2/0 mouse myeloma cells by using hybridoma-monoclonal antibody technology. Antibody titer, sensitivity and specificity of monoclonal antibodies were further determined by ELISA method. The formaldehyde-inactivated largemouth bass Largemouth bass ranavirus (LMBV) vaccine was prepared to immunize largemouth bass, and the antibody level after immunization was detected by the prepared monoclonal antibody. Two monoclonal antibody hybridoma cell line named A9E7and C9B9 were obtained, and ascites antibody titer of A9E7and C9B9 were reached up to 1:2.187×106 and 1:7.29×105, respectively. The measuring sensitivity of the A9E7 to purified IgM was as low as 10 ng, and the corresponding sensitivity of C9B9 was 20 ng. The results of cross reaction showed that both A9E7 and C9B9 binded specifically to the immunoglobulin of largemouth bass, A9E7 binded to the serum immunoglobulin of sea perch Lateolabrax maculatus, and C9B9 binded to mandarinfish Siniperca chuatsi. However, no cross reaction was found with grass carp Ctenopharyngodon idellus, crucian carp Carrassius auratus, loach Misgurnus anguillicaudatus, bluntnose black bream Megalobrama amblycephala, Acrossocheilus fasciatus, yellow catfish Pelteobagrus fulvidraco, topmouth culter Culter alburnus, ricefield Monopterus albus, silver carp Hypophthalmichthys molitrix and bighead carp Aristichthys nobilis. After immunizing the LMBV, the antibody level in largemouth bass was increased significantly with peak at 35 d, and with antibody titer of 1:1280. Monoclonal antibody against immunoglobulin of largemouth bass was prepared with strong specificity and high sensitivity, which can be used for early disease monitoring, immune response regularity and vaccine development of largemouth bass.

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大口黑鲈免疫球蛋白单克隆抗体的制备及初步应用