| 三疣梭子蟹颗粒血细胞单克隆抗体的研制及7种甲壳类动物血细胞的交叉反应研究 |
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| 引用本文： | 王颖,王文琪,程顺峰.三疣梭子蟹颗粒血细胞单克隆抗体的研制及7种甲壳类动物血细胞的交叉反应研究[J].水产学报,2015,39(6):810-817. |
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| 作者姓名： | 王颖 王文琪 程顺峰 |
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| 作者单位： | 青岛农业大学海洋科学与工程学院, 山东 青岛 266109,青岛农业大学海洋科学与工程学院, 山东 青岛 266109,青岛农业大学海洋科学与工程学院, 山东 青岛 266109 |
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| 基金项目： | 山东省“两区”建设专项;山东省现代农业产业技术体系虾蟹类创新团队项目(SDAIT-15-011-06) |
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| 摘 要： | 为研究虾蟹类甲壳动物血细胞膜表面是否具有相同的抗原决定簇及共同抗原表位的生物学特征,采用制备的抗三疣梭子蟹颗粒血细胞单克隆抗体,通过激光共聚焦扫描显微镜(laser scanning confocal microscopy,LSCM)、免疫印迹(Western blot)和流式细胞术(Flow Cytometry,FCM)等多种方法,测定了7种甲壳类动物(凡纳滨对虾、中国明对虾、刀额新对虾、口虾蛄、中华绒螯蟹、日本板蟹及美洲黄道蟹)颗粒血细胞及透明血细胞与三疣梭子蟹颗粒血细胞单克隆抗体发生特异性结合的抗原表位。LSCM可观察到该株抗三疣梭子蟹颗粒血细胞单克隆抗体与中华绒螯蟹和日本板蟹血细胞交叉反应结果为阳性,分析发现在中华绒螯蟹血淋巴中阳性颗粒血细胞在其颗粒血细胞总数中占76.74%,阳性透明血细胞在其透明血细胞总数中占70.59%,日本板蟹阳性颗粒血细胞在其颗粒血细胞总数中占73.86%,阳性透明血细胞在其透明血细胞总数中占16.67%,其余5种甲壳类动物均为阴性;Western blot测试结果显示该株单克隆抗体仅与中华绒螯蟹血细胞反应,且发生反应的抗原决定簇位于分子量为30 ku的蛋白带上;FCM分析发现该株单克隆抗体与中华绒螯蟹透明血细胞和颗粒血细胞均可发生交叉反应,阳性率分别为57.72%和77.05%,与日本板蟹透明血细胞阳性反应极少,阳性率仅为9.57%,与颗粒血细胞发生阳性反应的阳性率为82.59%。
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| 关 键 词： | 三疣梭子蟹 单克隆抗体 颗粒血细胞 交叉反应 |
| 收稿时间： | 1/2/2015 12:00:00 AM |
| 修稿时间： | 3/1/2015 12:00:00 AM |
Antigenic cross-reactivity of crustacean haemocytes using monoclonal antibodies produced against granulocyte of the swimming crab(Portunus trituberculatus) |
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| WANG Ying,WANG Wenqi and CHENG Shunfeng.Antigenic cross-reactivity of crustacean haemocytes using monoclonal antibodies produced against granulocyte of the swimming crab(Portunus trituberculatus)[J].Journal of Fisheries of China,2015,39(6):810-817. |
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| Authors: | WANG Ying WANG Wenqi and CHENG Shunfeng |
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| Institution: | College of Marine Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China,College of Marine Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China and College of Marine Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China |
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| Abstract: | Using the monoclonal antibodies(Mab)3F4 against granulocyte of swimming crab(Portunus trituberculatus)to research the cross reaction of other seven species of crustaceans(Penaeus vannamei,Fenneropenaeus chinensis,Metapenaeus ensis,Oratosquilla oratoria,Eriocheir sinensis,Petalomera japonica,Cancer magister).In this paper,laser scanning confocal microscopy(LSCM),Western blot and Flow Cytometry(FCM)were employed to detect the relationship between these seven species.Balb/C mice were immunized by heamocytes of Portunus trituberculatus.Spleens were dissected from the immunized Balb/c mice and the cells were fused with SP2/0 myeloma cell line using polyethylene glycol as fusogen.Hybridomaculture mediums of hybridoma cells were selected using Indirect Immunofluorescence Assay(IFA)screening methods.One positive hybridoma was found which secreted high titer antibody,followed by single-cell cloning and subcloning by limiting dilution method and tested again for their specificity by IFA at the appropriate stage of growth.Single-cell cloning was repeated at least twice so as to prove the monoclonality of the hybridoma culture supernatant.We got the MAb 3F4 specific against granulocyte of swimming crab(Portunus trituberculatus).MAb 3F4 showed strong immunofluorescent reaction with granulocytes,but no reaction with hyalinocytes.In cross-reactivity,LSCM showed the MAb 3F4 could also react with haemocytes of Eriocheir sinensis and Petalomera japonica. Sorted by FCM,MAb 3F4 was positive to the two types of heamocytes of Eriocheir sinensis(hyalinocyte and granulocyte),and the percentage of positive rate were 57.72% and 77.05% respectively.Reacting with haemocyte of Petalomera japonica was mainly positive to granulocyte,whose positive rate was 82.59%,but that to the hyalinocyte was only 9.57%.Analyzed by Western blot,MAb 3F4 was recognized as one heamocyte protein of Eriocheir sinensis,whose molecule weight was 30 ku,but there was no protein band on nitrocellulose membrane of Petalomera japonica. |
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| Keywords: | Portunus trituberculatus monoclonal antibody granulocyte cross reaction |
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