抗鸡CD8α单克隆抗体的研制与初步应用
摘要
单克隆抗体一直以来都被作为研究T淋巴细胞表面分子的重要工具。国外研制的鸡CD分子的单克隆抗体为研究免疫细胞的生理功能及细胞表面标志的生物学作用等创造了有利条件,他们可以利用这些单克隆抗体对淋巴细胞进行分选,以研究免疫应答类型;或用于消耗动物体内相应的CD分子,以研究CD分子在体内的作用;也可用于研究CD分子的结构、功能等。本实验旨在研制特异性的抗鸡CD8α的单克隆抗体,特别是适用于间接免疫荧光试验、细胞分析与分选等流式细胞术的单克隆抗体。
研究中首先利用淋巴细胞分离液制备白莱航鸡脾脏和胸腺的淋巴细胞,接着用制备的淋巴细胞免疫3只6周龄的雌性BALB/c小鼠。在经过两次免疫后,眼静脉窦采血,分离血清,应用间接免疫荧光试验测定血清效价。达到1:1280后,进行加强免疫。3天后,无菌条件下取BALB/c小鼠的脾脏细胞与培养到对数生长期的小鼠骨髓瘤细胞系SP2/0在PEG4000的作用下进行融合。5天后添加新鲜的HAT培养基,10天后更换HT培养基。待融合细胞上清变黄后即可进行阳性克隆的筛选。
采用间接免疫荧光染色结合FACS分析筛选阳性克隆。应用本室构建的稳定表达鸡CD8α的细胞系Flp-in-293-CD8α-FLAG与杂交瘤上清进行间接免疫荧光染色,通过流式细胞术检测荧光率筛选出阳性杂交瘤克隆。同时设立商品化的纯化的鼠抗鸡CD8α抗体的阳性对照。再经过2次有限稀释法亚克隆,筛选到了一株稳定分泌抗鸡CD8α的单克隆抗体的杂交瘤细胞,命名为6E5。将杂交瘤细胞6E5腹腔注射预先用石蜡处理的BALB/c小鼠,制备6E5腹水。间接免疫荧光FACS试验测定腹水效价,达到1:1204000。应用抗原介导的ELISA方法,测得6E5的亚类为IgG1。
应用CD3/CD8双色免疫荧光结合FACS分析进一步鉴定单克隆抗体。取白莱航鸡的脾脏淋巴细胞,第一步通过间接染色的方法标记6E5抗鼠FITC,第二步通过直接染色的方法标记CD3-PE。同时设立商品化的纯化的鼠抗鸡CD8α抗体为一抗的阳性对照。结果显示,6E5是一株分泌抗鸡CD8α的单克隆抗体的杂交瘤细胞。
将6E5与商品化的鼠抗鸡CD8α抗体(CLONE:CT8)进行抗原表位的比较。取白莱航鸡的淋巴细胞,加入不同含量的6E5杂交瘤细胞制备的腹水,接着加入相应含量的CD8-PE(CLONE:CT8)。如果6E5与商品化的鼠抗鸡CD8α抗体(CLONE:CT8)是识别相同的表位,那么随着6E5腹水量的升高,荧光率应该降低。但是实验的结果表明,荧光率不随着6E5浓度的升高而降低,说明两者不是针对同一抗原表位。
应用单克隆抗体6E5测定与白莱航品系鸡不同组织中淋巴细胞的反应性。取白莱航鸡的胸腺、脾脏、法氏囊淋巴细胞,与6E5腹水分别反应。结果表明,6E5可以识别胸腺、脾脏淋巴细胞里表达CD8 T淋巴细胞。但是不与法氏囊淋巴细胞反应,这主要是因为法氏囊淋巴细胞是B淋巴细胞。
应用单克隆抗体6E5测定与不同品种(系)的家禽的淋巴细胞的反应性。选取隐性白羽鸡、石岐杂鸡、狼山鸡、安卡鸡、龙溪麻鸡、康贝尔鸭、鸦鸭、扬州鹅八种家禽,分别采集外周血,制备外周血淋巴细胞,测定6E5与不同品系的家禽的淋巴细胞的反应性。结果显示,6E5可以特异的识别鸡的外周血淋巴细胞中表达CD8的T淋巴细胞,不能识别康贝尔鸭、鸦鸭、扬州鹅外周血淋巴细胞。
在鸡的免疫试验中,进一步应用单克隆抗体6E5测定了不同免疫组的鸡的免疫水平。
利用本室构建的猪霍乱沙门菌C500△asd(pYA3334)菌株、猪霍乱沙门菌C500△asd(pYA3334-F)菌株免疫20日龄海兰白鸡,同时设立不免疫的阴性对照。在第一次免疫后14天、第二次免疫后7天、第二次免疫后14天,分别取不同免疫组的海兰白鸡,制备脾脏淋巴细胞,利用间接方法标记6E5抗鼠PE。再标记CD4-FITC,流式细胞仪检测CD4与CD8百分含量。同时设立商品化鼠抗鸡CD8α抗体的阳性对照。通过CD4与CD8的百分含量的比值判断不同免疫组的白莱航鸡的免疫水平。6E5测得的结果与商品化的鼠抗鸡CD8α抗体得到的结果相同。说明6E5是一株可以应用于流式细胞术检测的单克隆抗体。
Monoclonal antibodies (mAbs) against the surface molecules including CDs of T lymphocytes are valuable tools to study the development and function of distinct subpopulations of chicken T lymphocytes, to purify lymphocytes in order to analyze immune responses, to define the role of CD molecules after blocking these molecules based on mAbs in vivo, to analyze the structure and function of CD molecules. In the present study, the specific monoclonal mAb against chicken CD8α(ChCD8α) was developed.
Three six-week old female BALB/c mice were immunized intraperitoneally with 1×106 lymphocytes of White Leghorn chicken every two weeks interval. Three days prior to fusion, mice were boosted with five times of the immunization dosage .Then the mice were sacrificed and splenocytes were harvested. Two weeks after fusion, culture supernatants of hybrid cells were screened by indirect immunofluorescence FACS using the cell line Flp-in-293-CD8α-flag as detecting immunogen.
Positive hybrids were subcloned twice by limiting dilution to ensure that antibody producing cells were truly monoclonal and that the antibody secretion could be stable. One positive clone named 6E5. The hybridoma 6E5 was injected into pristine-primed mice (approximately 1×106 hybridoma cells/mouse) for ascite production. And then the subclass of mAb were determined as IgG1 by antigen-mediated ELISA employing. The titers of mAb was up to 1:1204000 in indirect immunofluorescent FACS assay.
Using CD3/CD8 double staining FACS analysis, the results of 6E5 reacted with lymphocytes of White Leghorn chicken was coincided well with those of commercial mAb CT8. It suggests that 6E5 is a specific mAb against chicken CD8α.
The epitopes recognized by 6E5 and commercial mAb CT8 were evaluated by FACS with the competitive immunological staining. It was shown that there was no inhibition between 6E5 and CT8 to binding lymphocytes White Leghorn chicken. The results demonstrated that the recognized epitopes were different for both of two mAbs.
The specificity of mAb 6E5 was determined through detecting the reactivity with lymphocytes from different lymph tissues, including thymus, spleen and bursa of White Leghorn chicken. The results showed that mAb 6E5 could react with CD8+ T cells from thymus and spleen, but not bind to lymphocytes of bursa, which belong to B cells.
The reactivity spectrum of mAb 6E5 was further evaluated to detecting the binding with peripheral blood mononuclear cells(PBMC) from different species of poultry, including Recessive White chicken, Shack-kee chicken, Langshan chicken, Anka chicken, Longxi partridge chicken, Campbell duck, Crow duck, Yangzhou goose. The results showed that mAb 6E5 could react with PBMC from all chicken species tested, but not with those from duck and goose. These results further demonstrated that mAb 6E5 was a useful antidody with good reactivity and specificity in FACS.
Moreover, a FACS method based on mAb 6E5 was develop to measure the ratio of CD4+ lymphocytes with CD8+ ones. Hy-line White layer were immunized with Salmonella choleraesuis C500△asd(pYA3334) and Salmonella choleraesuis C500△asd(pYA3334-F), respectively. The ratio of CD4+ lymphocytes with CD8+ ones was monitored at different time points in different immunized groups. The results were coincided well between both 6E5 and CT8.
All these results suggested that mAb 6E5 has been successfully developed, and is a very useful reagent in further studies.
研究中首先利用淋巴细胞分离液制备白莱航鸡脾脏和胸腺的淋巴细胞,接着用制备的淋巴细胞免疫3只6周龄的雌性BALB/c小鼠。在经过两次免疫后,眼静脉窦采血,分离血清,应用间接免疫荧光试验测定血清效价。达到1:1280后,进行加强免疫。3天后,无菌条件下取BALB/c小鼠的脾脏细胞与培养到对数生长期的小鼠骨髓瘤细胞系SP2/0在PEG4000的作用下进行融合。5天后添加新鲜的HAT培养基,10天后更换HT培养基。待融合细胞上清变黄后即可进行阳性克隆的筛选。
采用间接免疫荧光染色结合FACS分析筛选阳性克隆。应用本室构建的稳定表达鸡CD8α的细胞系Flp-in-293-CD8α-FLAG与杂交瘤上清进行间接免疫荧光染色,通过流式细胞术检测荧光率筛选出阳性杂交瘤克隆。同时设立商品化的纯化的鼠抗鸡CD8α抗体的阳性对照。再经过2次有限稀释法亚克隆,筛选到了一株稳定分泌抗鸡CD8α的单克隆抗体的杂交瘤细胞,命名为6E5。将杂交瘤细胞6E5腹腔注射预先用石蜡处理的BALB/c小鼠,制备6E5腹水。间接免疫荧光FACS试验测定腹水效价,达到1:1204000。应用抗原介导的ELISA方法,测得6E5的亚类为IgG1。
应用CD3/CD8双色免疫荧光结合FACS分析进一步鉴定单克隆抗体。取白莱航鸡的脾脏淋巴细胞,第一步通过间接染色的方法标记6E5抗鼠FITC,第二步通过直接染色的方法标记CD3-PE。同时设立商品化的纯化的鼠抗鸡CD8α抗体为一抗的阳性对照。结果显示,6E5是一株分泌抗鸡CD8α的单克隆抗体的杂交瘤细胞。
将6E5与商品化的鼠抗鸡CD8α抗体(CLONE:CT8)进行抗原表位的比较。取白莱航鸡的淋巴细胞,加入不同含量的6E5杂交瘤细胞制备的腹水,接着加入相应含量的CD8-PE(CLONE:CT8)。如果6E5与商品化的鼠抗鸡CD8α抗体(CLONE:CT8)是识别相同的表位,那么随着6E5腹水量的升高,荧光率应该降低。但是实验的结果表明,荧光率不随着6E5浓度的升高而降低,说明两者不是针对同一抗原表位。
应用单克隆抗体6E5测定与白莱航品系鸡不同组织中淋巴细胞的反应性。取白莱航鸡的胸腺、脾脏、法氏囊淋巴细胞,与6E5腹水分别反应。结果表明,6E5可以识别胸腺、脾脏淋巴细胞里表达CD8 T淋巴细胞。但是不与法氏囊淋巴细胞反应,这主要是因为法氏囊淋巴细胞是B淋巴细胞。
应用单克隆抗体6E5测定与不同品种(系)的家禽的淋巴细胞的反应性。选取隐性白羽鸡、石岐杂鸡、狼山鸡、安卡鸡、龙溪麻鸡、康贝尔鸭、鸦鸭、扬州鹅八种家禽,分别采集外周血,制备外周血淋巴细胞,测定6E5与不同品系的家禽的淋巴细胞的反应性。结果显示,6E5可以特异的识别鸡的外周血淋巴细胞中表达CD8的T淋巴细胞,不能识别康贝尔鸭、鸦鸭、扬州鹅外周血淋巴细胞。
在鸡的免疫试验中,进一步应用单克隆抗体6E5测定了不同免疫组的鸡的免疫水平。
利用本室构建的猪霍乱沙门菌C500△asd(pYA3334)菌株、猪霍乱沙门菌C500△asd(pYA3334-F)菌株免疫20日龄海兰白鸡,同时设立不免疫的阴性对照。在第一次免疫后14天、第二次免疫后7天、第二次免疫后14天,分别取不同免疫组的海兰白鸡,制备脾脏淋巴细胞,利用间接方法标记6E5抗鼠PE。再标记CD4-FITC,流式细胞仪检测CD4与CD8百分含量。同时设立商品化鼠抗鸡CD8α抗体的阳性对照。通过CD4与CD8的百分含量的比值判断不同免疫组的白莱航鸡的免疫水平。6E5测得的结果与商品化的鼠抗鸡CD8α抗体得到的结果相同。说明6E5是一株可以应用于流式细胞术检测的单克隆抗体。
Monoclonal antibodies (mAbs) against the surface molecules including CDs of T lymphocytes are valuable tools to study the development and function of distinct subpopulations of chicken T lymphocytes, to purify lymphocytes in order to analyze immune responses, to define the role of CD molecules after blocking these molecules based on mAbs in vivo, to analyze the structure and function of CD molecules. In the present study, the specific monoclonal mAb against chicken CD8α(ChCD8α) was developed.
Three six-week old female BALB/c mice were immunized intraperitoneally with 1×106 lymphocytes of White Leghorn chicken every two weeks interval. Three days prior to fusion, mice were boosted with five times of the immunization dosage .Then the mice were sacrificed and splenocytes were harvested. Two weeks after fusion, culture supernatants of hybrid cells were screened by indirect immunofluorescence FACS using the cell line Flp-in-293-CD8α-flag as detecting immunogen.
Positive hybrids were subcloned twice by limiting dilution to ensure that antibody producing cells were truly monoclonal and that the antibody secretion could be stable. One positive clone named 6E5. The hybridoma 6E5 was injected into pristine-primed mice (approximately 1×106 hybridoma cells/mouse) for ascite production. And then the subclass of mAb were determined as IgG1 by antigen-mediated ELISA employing. The titers of mAb was up to 1:1204000 in indirect immunofluorescent FACS assay.
Using CD3/CD8 double staining FACS analysis, the results of 6E5 reacted with lymphocytes of White Leghorn chicken was coincided well with those of commercial mAb CT8. It suggests that 6E5 is a specific mAb against chicken CD8α.
The epitopes recognized by 6E5 and commercial mAb CT8 were evaluated by FACS with the competitive immunological staining. It was shown that there was no inhibition between 6E5 and CT8 to binding lymphocytes White Leghorn chicken. The results demonstrated that the recognized epitopes were different for both of two mAbs.
The specificity of mAb 6E5 was determined through detecting the reactivity with lymphocytes from different lymph tissues, including thymus, spleen and bursa of White Leghorn chicken. The results showed that mAb 6E5 could react with CD8+ T cells from thymus and spleen, but not bind to lymphocytes of bursa, which belong to B cells.
The reactivity spectrum of mAb 6E5 was further evaluated to detecting the binding with peripheral blood mononuclear cells(PBMC) from different species of poultry, including Recessive White chicken, Shack-kee chicken, Langshan chicken, Anka chicken, Longxi partridge chicken, Campbell duck, Crow duck, Yangzhou goose. The results showed that mAb 6E5 could react with PBMC from all chicken species tested, but not with those from duck and goose. These results further demonstrated that mAb 6E5 was a useful antidody with good reactivity and specificity in FACS.
Moreover, a FACS method based on mAb 6E5 was develop to measure the ratio of CD4+ lymphocytes with CD8+ ones. Hy-line White layer were immunized with Salmonella choleraesuis C500△asd(pYA3334) and Salmonella choleraesuis C500△asd(pYA3334-F), respectively. The ratio of CD4+ lymphocytes with CD8+ ones was monitored at different time points in different immunized groups. The results were coincided well between both 6E5 and CT8.
All these results suggested that mAb 6E5 has been successfully developed, and is a very useful reagent in further studies.
引文
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[1]Chan MM, Chen CL, Ager LL, et al. Identification of the avian homologues of mammalian CD4 and CD8 antigens. Immunlo,1988,140:2133-2138.
[2]Luhtala M, Koskinen MR, Toivanen P. Characterization of chicken CD8-specific monoclonal antibodies recognizing novel epitopes. Immunol, 1995, 42:171-174.
[3]Luhtala M. Chicken CD4, CD8αβ, and CD8ααT Cell Co-Receptor Molecules. Poultry Science 1998, 77:1858-1873
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[5]Cihak J, Hoffmann-Fezer G, Wasl M, et al. Inhibition of the development of spontaneous autoimmune thyroiditis in the obese strain (OS) chickens by in vivo treatment with anti-CD4 or anti-CD8 antibodies. J Autoimmun, 1998, 11(2): 119-126.
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[10]胡青海.鸡IL-2、IL-18、IFN-γ和CpG DNA在减毒沙门氏菌运送H5亚型禽流感核酸疫苗中的佐剂作用及鸡CD4和CD8分子单克隆抗体的研制.扬州大学博士学位论文, 2005.
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[1]Chan MM, Chen CL, Ager LL, et al. Identification of the avian homologues of mammalian CD4 and CD8 antigens. Immunlo,1988,140:2133-2138.
[2]Luhtala M, Koskinen MR, Toivanen P. Characterization of chicken CD8-specific monoclonal antibodies recognizing novel epitopes. Immunol, 1995, 42:171-174.
[3]Luhtala M. Chicken CD4, CD8αβ, and CD8ααT Cell Co-Receptor Molecules. Poultry Science 1998, 77:1858-1873
[4]刘秀梵,主编.单克隆抗体在农业上的应用.安徽科学技术出版社,1994.
[5]Cihak J, Hoffmann-Fezer G, Wasl M, et al. Inhibition of the development of spontaneous autoimmune thyroiditis in the obese strain (OS) chickens by in vivo treatment with anti-CD4 or anti-CD8 antibodies. J Autoimmun, 1998, 11(2): 119-126.
[6]Valdez RA, McGuire TC, Brown WC, et al. Long-term in vivo depletion of functional CD4+ T lymphocytes from calves requires both thymectomy and anti-CD4 monoclonal antibody treatment. Immunology,2001, 102 :426-433.
[7]胡青海,焦新安.鸡CD4和CD8分子研究进展.动物医学进展, 2005, 26(4): 16-20.
[8]Leinonen J, Niemel? P, L?vgren J, et al. Characterization of monoclonal antibodies against prostate specific antigen produced by genetic immunization. J Immunol Methods, 2004, 289(1-2): 157-167.
[9]Moonsom S, Khunkeawla P, Kasinrerk W. Production of polyclonal and monoclonal antibodies against CD54 molecules by intrasplenic immunization of plasmid DNA encoding CD54 protein. Immunology Letters, 2001,76 :25–30
[10]胡青海.鸡IL-2、IL-18、IFN-γ和CpG DNA在减毒沙门氏菌运送H5亚型禽流感核酸疫苗中的佐剂作用及鸡CD4和CD8分子单克隆抗体的研制.扬州大学博士学位论文, 2005.
[11]Kothlow S, Mannes NK, Schaerer B, et al. Characterization of duck leucocytes by monoclonal antibodies. Dev Comp Immunol, 2005, 29(8): 733-748.
[12]Attanasio R, Pehler K, Scinicariello F. DNA-based immunization induces anti-CD4 antibodies directed primarily to native epitopes. FEMS Immunol Med Microbiol,1997, 17(4): 207-215.
[13]樊一笋.两株抗人CD40配体单克隆抗体的制备及其生物学功能研究.苏州大学硕士学位论文, 2001.
[14]袁舟.稳定表达鸡CD4或CD8α细胞的构建.扬州大学硕士学位论文, 2008.
[15]Breed DGJ, Carr P, Vermeulen AN. Differential binding of two monoclonal antibodies directed against the chicken CD8αmolecule. Veterinary Immunology and Immunopathology, 1996, 52:117-125.
[16]邱玉华. B7:CD28激发/拮抗型单抗的研制及其在共刺激信号传导中的调节作用研究苏州大学博士学位论文, 2001.
[17]Masayuki S, Marcelo RP, Maeda K, et al. Characterization of anti-feline CD8 monoclonal antibodies. Veternary immunology and immunopathology ,1998,61:17-23.
[18]文飚.鼠抗人B7-1分子功能性单克隆抗体的研制及其生物学特性的研究.苏州大学硕士学位论文, 2002.
[19]Fan Y, Ge Y, Zhu H, et al. Characterization and application of two novel monoclonal antibodies against CD40L:epitope and functional studies on cell membrane CD40L and studies on cell membrane CD40L and studies on the origin of soluble serum CD40L. Tissue Antigens, 2004,64:257-263.
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