重组表达了牙鲆(Paralichth ys olivaceus)T淋巴细胞表面标志分子CD3的蛋白,制备出其兔多克隆抗体(多抗),采用免疫双荧光流式细胞术,观察外周血白细胞中的T、B淋巴细胞并计算其比例.分别向牙鲆腹腔内注射107 CFU/mL的迟缓爱德华氏菌和其灭活疫苗,于0、1、3、5、7、9、14、21和28天后抽取外周血,用抗红细胞单抗的流式细胞术测定红细胞和白细胞的比例和浓度,提取外周血白细胞,用流式细胞术测定T、B淋巴细胞的比例.研究显示,CD3多抗和IgM单抗分别识别T、B淋巴细胞,二抗体无交叉反应,白细胞中T细胞占(7.49±1.5)％、B细胞占(14.64±1.8)％.感染和免疫后,牙鲆外周血红细胞浓度变化不显著,为(2.9±0.45)×109 cells/mL.感染组,白细胞浓度第3天显著上升,第14天达到最大值(4.7±1.7)×108 cells/mL;T细胞比例第1天显著上升,第9天达到最大值(24.3±1.28)％;B细胞比例第1天显著升高,第21天达到最大值(40.9±1.52)％.免疫组,白细胞浓度第3天显著上升,第14天达最大值(7.1±1.8)×108 cells/mL;T细胞比例第5天显著上升,第9天达最大值(20.5±1.12)％;B细胞比例第1天显著上升,第21天达到最大值(39.3±1.55)％.对照组,白细胞浓度为(6.9±1.6)× 107 cells/mL,T细胞比例为(8.05±1.38)％,B细胞比例为(13.77±1.56)％.研究结果表明,感染和免疫后牙鲆白细胞浓度、T、B细胞的比例均显著升高,感染组T、B细胞比例显著高于免疫组.本研究结果为血细胞作为牙鲆健康评估指标提供了数据资料.%Fish blood cells involve in the process of immune response and anti-infection.The leukocytes,especially T and B lymphocytes,play important roles in fish immunity,and in the meantime,the number of blood cells or lymphocytes vary during the physiologic processes.In this study,the variation of peripheral blood cells of flounder (Paralichthys olivaceus) after Edwardsiella tarda infection and vaccination was determined.Our primary research has produced anti-erythrocytes monoclonal antibody (Mab) and anti-immunoglobulin (IgM) Mab,respectively.In this study,the CD3 molecule was cloned and its recombined protein was expressed,and then rabbit anti-CD3 polyclonal antibody (RaPoCD3-Pab) was produced.T and B lymphocytes in peripheral blood (PBL) cells were observed and counted by flow cytometry (FCM) using RaPoCD3-Pab and IgM Mab,respetively.Flounder was intraperitoneally injected with 1× 107CFU/mL Edwardsiella tarda,its inactivated vaccine and PBS,respectively.The PBL was collected on days 0,1,3,5,7,9,14,21 and 28 post injection,respectively.Erythrocytes and leukocytes were analyzed by double fluorescent FCM using Mab against erythrocytes of flounder,and T and B lymphocytes were counted by double fluorescent FCM with RaPoCD3-Pab and IgM Mab.The result showed that the RaPoCD3-Pab and IgM Mab specifically recognized T and B lymphocytes,which were labeled by red or green fluorescence,respectively,and no cross reaction was detected.The T cells were in percentage of (7.49±1.5)％ and B cells were (14.64±1.8)％ in leukocytes.After infection or vaccination,the concentration of erythrocytes had no significant variation (P＜0.01,ANOVA) during the whole experiment compared with the control group,keeping at (2.9±0.45) × 109 cells/mL in PBL.The leukocyte concentration in control group maintained at (6.9±1.6) × 107 cells/mL,the proportion of T cells was (8.05±1.38)％,the proportion of B cells was (13.77±1.56)％.In the infection group,the concentration of leukocyte increased significantly on the 3rd day,then reached the peak at (4.7 ± 1.7) × 108cells/mL on the 14th day.The proportion of T cells rose immediately on the 1st day,then reached the maximum of (24.3±1.28)％ on the 9th day.The proportion of B cells increased on the 1st day,and reached the peak (40.9±1.52) ％ on the 21st day (P＜0.01,ANOVA).In the vaccination group,the leukocyte concentration increased significantly on the 3rd day,then reached the peak of (7.1±1.8)× 108 cells/mL on the 14th day.The proportion of T cells rose significantly since the 5th day,then reached the maximum of (20.5± 1.12)％ on the 9th day.The proportion of B cells increased on the 1st day,and reached the peak of (39.3 ±1.55) ％ on the 21st day (P＜0.01,ANOVA).The results indicated that leukocyte concentration,the proportion of T and B cells in PBL increased significantly before the 21th day after infection and vaccination,leukocyte concentration kept rising during a period of 3-14 days,T cells percentage rose till the 9th day,B cells percentage climbed up till the 21th day,the proportions of T and B cells in PBL increased significantly after infection and vaccination,especiallyon the 1st day.The counts of leukocytes or the percentage of T and B cells varied with the infection or vaccination process,which might be potential indexes for fish physiologic processes.
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