茄科劳尔氏菌单克隆抗体的制备及其特性鉴定
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摘要
茄科劳尔氏菌(Ralstonia solanacearum,RS)是番茄、茄子、辣椒、马铃薯等茄科蔬菜青枯病害的致病菌。为实现对RS的快速高效检测,以茄科劳尔氏菌株1.76免疫BALB/c小鼠,经细胞融合后利用间接酶联免疫吸附分析(Enzyme-linked Immunosorbent Assay,ELISA)筛选出3株能稳定分泌抗茄科劳尔氏菌株1.76的单克隆杂交瘤细胞株1C1、1B3和9D7。然后利用小鼠体内诱生腹水,1C1、1B3和9D7效价分别为1:1 024 000、1:64 000、1:256 000。采用饱和硫酸铵沉淀及Protein-G亲和层析法纯化腹水,经SDS-PAGE鉴定显示纯化后的单克隆抗体(Monoclonal Antibodies,mAb)纯度较高。纯化后单克隆抗体(2 mg/mL)效价分别为1:17 529、1:35 819、1:50 000,抗体亚型均为IgG1。对3株抗体进行特异性检测结果显示,1C1和9D7均不能与RS-5结合,1B3不能结合1.74和RS-5。此外,检测结果还表明3株单克隆抗体与桑肠杆菌JX-6、苏云金芽胞杆菌SYJ及实验室现有11株燕麦嗜酸菌卡特莱兰亚种、燕麦嗜酸菌西瓜亚种、玉米细菌性条斑菌、嗜酸菌魔芋亚种,梨火疫病菌QB0809、 XL-4,玉米细菌性枯萎病菌QB0241、QB0242,水稻细菌性谷枯病菌QB0017、QB0753、QB0755均无交叉情况。此次茄科劳尔氏菌抗体的制备,为后期青枯病菌的快速检测提供参考。
Ralstonia solanacearum(RS) is a pathogen that causes tomato, eggplant, pepper, and other vegetables green wilt. In order to achieve a rapid and efficient detection of RS, BALB/c mice were immunized with RS strain 1.76 in this study, three hybridoma strains, 1 C1, 1 B3 and 9 D7, that could stably secret monoclonal antibodies(mAbs) against the RS strain 1.76, was screened after cell fusion and analyzed by indirect ELISA. Then using induced mice intrinsic ascites, and tested the valences of 1 C1, 1 B3 and 9 D7 were at 1:1 024 000, 1:64 000 and 1:256 000, respectively. The ascites was purified by saturated ammonium sulfate precipitation and Protein-G affinity chromatography, after the purification the purity of mAb was characterized by SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis) showed the mAb was at high purity. After the purification, the valences of mAbs(2 mg/mL) were at 1:17 529, 1:35 819 and 1:50 000, respectively. And the subtypes of mAbs were all IgG1. The results of three antibodies specificity test have shown that neither 1 C1 nor 9 D7 could bind to RS-5, and 1 B3 could not bind to 1.74 and RS-5. In addition, the test results also showed that the three mAbs had no cross-reaction with Enterobacter mori JX-6, Bacillus thuringiensis SYJ and all the laboratory existing 11 strains of Acidovorax avenae subsp. citrulli, Acidovorax cattleyae, Acidovorax konjaci, Acidovorax avenae, pear fire blight Erwinia amylovora QB0809, XL-4, corn bacterial blight pathogen Pantoea stewartii subsp. stewartii QB0241, QB0242, and paddy bacterial glume blight pathogen Burkholderia glumae QB0017, QB0753, QB0755. The preparation of Ralstonia solanacearum antibody in this study had laid the foundation for rapid determination of bacterial wilt pathogens later.
Ralstonia solanacearum(RS) is a pathogen that causes tomato, eggplant, pepper, and other vegetables green wilt. In order to achieve a rapid and efficient detection of RS, BALB/c mice were immunized with RS strain 1.76 in this study, three hybridoma strains, 1 C1, 1 B3 and 9 D7, that could stably secret monoclonal antibodies(mAbs) against the RS strain 1.76, was screened after cell fusion and analyzed by indirect ELISA. Then using induced mice intrinsic ascites, and tested the valences of 1 C1, 1 B3 and 9 D7 were at 1:1 024 000, 1:64 000 and 1:256 000, respectively. The ascites was purified by saturated ammonium sulfate precipitation and Protein-G affinity chromatography, after the purification the purity of mAb was characterized by SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis) showed the mAb was at high purity. After the purification, the valences of mAbs(2 mg/mL) were at 1:17 529, 1:35 819 and 1:50 000, respectively. And the subtypes of mAbs were all IgG1. The results of three antibodies specificity test have shown that neither 1 C1 nor 9 D7 could bind to RS-5, and 1 B3 could not bind to 1.74 and RS-5. In addition, the test results also showed that the three mAbs had no cross-reaction with Enterobacter mori JX-6, Bacillus thuringiensis SYJ and all the laboratory existing 11 strains of Acidovorax avenae subsp. citrulli, Acidovorax cattleyae, Acidovorax konjaci, Acidovorax avenae, pear fire blight Erwinia amylovora QB0809, XL-4, corn bacterial blight pathogen Pantoea stewartii subsp. stewartii QB0241, QB0242, and paddy bacterial glume blight pathogen Burkholderia glumae QB0017, QB0753, QB0755. The preparation of Ralstonia solanacearum antibody in this study had laid the foundation for rapid determination of bacterial wilt pathogens later.
引文
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[2] He LY. Characteristics of Strains of Pseudomonas solanacearum from China[J]. Plant Disease, 1964,67(12):1357-1361.
[3] 单志慧,廖伯寿,谈宇俊,等.花生青枯菌潜伏侵染的酶联免疫血清检测技术[J]. 中国油料作物学报, 1997,(2):45-47.
[4] 曹梦琪,包奇,杨采风,等. 基于qPCR技术快速检测青枯劳尔氏菌5号生理小种的方法[J]. 蚕业科学,2015,41(5):807-814.
[5] 曹梦琪,王旭东,王俊,等. 基于PMA-qPCR检测青枯菌5号生理小种活菌的方法[J]. 蚕业科学, 2015,41(6):1004-1010.
[6] 黄家权,晏立英,叶小文,等. 青枯菌定量检测方法的建立及其在花生中的应用[J]. 中国农业科学, 2011,44(1):58-66.
[7] 王华, 潘家荣, 熊汉国. 免疫分析技术在食品安全检测中的应用现状[J]. 食品科技, 2007,(6):34-37.
[8] Postmes TJ, Hout JCV, Saat G, et al. A radioimmunoassay study and comparison of seasonal variation in plasma triiodothyronine and thyroxine concentrations in normal healthy persons[J]. Clinica Chimica Acta, 1974,50(2):189-195.
[9] 王艳, 常润磊, 周旭东. 青枯病快速检测研究概况[J]. 桉树科技, 2012,29(2):53-58.
[10] 孙广瑞, 李小兵, 王哲. 制备分泌单克隆抗体杂交瘤细胞小鼠腹水的方法[J]. 中国现代医生, 2007,45(5):3-4.
[11] 萨姆布鲁克. 分子克隆实验指南[M].北京: 科学出版社,1992.
[12] Fasihi-Ramandi M, Nedjad-Moghaddam A, Arabsalmany F, et al. Prduction and characterization of monoclonal and polyclonal antibody against recombinant outer membrane protein[J]. Am J Immunol, 2014,10(1):56-62.
[13] Griep RA, Cvan T, Jrcmvan B, et al. Development of specific recombinant monoclonal antibodies against the lipopolysaccharide of Ralstonia solanacearum Race 3[J]. Phytopathology, 1998,88(8):795-803.
[14] 白利叶,范晓东,周泽扬,等.用于青枯劳尔氏菌快速检测的靶标内切葡聚糖酶抗体制备与检测试验[J]. 蚕业科学, 2016,(2):203-209.
[15] 蔡少华,王远程,肖小文,等.分泌抗植物青枯细菌菌系特异性单克隆抗体杂交瘤细胞株的建立[J]. 中国农业科学, 1986,19(2):92-93.
[16] 谢云陆,王远程.应用单克隆抗体免疫学技术检测种姜姜瘟病的初步研究[J].植物保护, 1991,17(5):23-24.
[17] 谢云陆,高德香,何礼远,等.应用单克隆抗体免疫荧光技术检测马铃薯青枯病潜伏侵染的初步研究[J]. 中国马铃薯, 1992,(1):2-6.
[18] Kaushal NA, Kaushal DC. Production and characterization of monoclonal antibodies against substrate specific loop region of Plasmodium falciparum lactate dehydrogenase[J]. Immunological Investigations, 2014,43(6):556-571.
[19] Liu BH, Chu KC, Yu FY. Novel monoclonal antibody-based sensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for detecting aflatoxin M1 in milk[J]. Food Control, 2016,66:1-7.
[20] Marcossilva L, Narimatsu Y, Halim A, et al. Characterization of binding epitopes of CA125 monoclonal antibodies[J]. Journal of Proteome Research, 2014,13(7):3349-3359.
[21] Kim SH, Kim YN, Truong TT, et al. Development of a monoclonal antibody specific to envelope domain III with broad-spectrum detection of all four dengue virus serotypes[J]. Biochemical & Biophysical Research Communications, 2016,473(4):894.
[2] He LY. Characteristics of Strains of Pseudomonas solanacearum from China[J]. Plant Disease, 1964,67(12):1357-1361.
[3] 单志慧,廖伯寿,谈宇俊,等.花生青枯菌潜伏侵染的酶联免疫血清检测技术[J]. 中国油料作物学报, 1997,(2):45-47.
[4] 曹梦琪,包奇,杨采风,等. 基于qPCR技术快速检测青枯劳尔氏菌5号生理小种的方法[J]. 蚕业科学,2015,41(5):807-814.
[5] 曹梦琪,王旭东,王俊,等. 基于PMA-qPCR检测青枯菌5号生理小种活菌的方法[J]. 蚕业科学, 2015,41(6):1004-1010.
[6] 黄家权,晏立英,叶小文,等. 青枯菌定量检测方法的建立及其在花生中的应用[J]. 中国农业科学, 2011,44(1):58-66.
[7] 王华, 潘家荣, 熊汉国. 免疫分析技术在食品安全检测中的应用现状[J]. 食品科技, 2007,(6):34-37.
[8] Postmes TJ, Hout JCV, Saat G, et al. A radioimmunoassay study and comparison of seasonal variation in plasma triiodothyronine and thyroxine concentrations in normal healthy persons[J]. Clinica Chimica Acta, 1974,50(2):189-195.
[9] 王艳, 常润磊, 周旭东. 青枯病快速检测研究概况[J]. 桉树科技, 2012,29(2):53-58.
[10] 孙广瑞, 李小兵, 王哲. 制备分泌单克隆抗体杂交瘤细胞小鼠腹水的方法[J]. 中国现代医生, 2007,45(5):3-4.
[11] 萨姆布鲁克. 分子克隆实验指南[M].北京: 科学出版社,1992.
[12] Fasihi-Ramandi M, Nedjad-Moghaddam A, Arabsalmany F, et al. Prduction and characterization of monoclonal and polyclonal antibody against recombinant outer membrane protein[J]. Am J Immunol, 2014,10(1):56-62.
[13] Griep RA, Cvan T, Jrcmvan B, et al. Development of specific recombinant monoclonal antibodies against the lipopolysaccharide of Ralstonia solanacearum Race 3[J]. Phytopathology, 1998,88(8):795-803.
[14] 白利叶,范晓东,周泽扬,等.用于青枯劳尔氏菌快速检测的靶标内切葡聚糖酶抗体制备与检测试验[J]. 蚕业科学, 2016,(2):203-209.
[15] 蔡少华,王远程,肖小文,等.分泌抗植物青枯细菌菌系特异性单克隆抗体杂交瘤细胞株的建立[J]. 中国农业科学, 1986,19(2):92-93.
[16] 谢云陆,王远程.应用单克隆抗体免疫学技术检测种姜姜瘟病的初步研究[J].植物保护, 1991,17(5):23-24.
[17] 谢云陆,高德香,何礼远,等.应用单克隆抗体免疫荧光技术检测马铃薯青枯病潜伏侵染的初步研究[J]. 中国马铃薯, 1992,(1):2-6.
[18] Kaushal NA, Kaushal DC. Production and characterization of monoclonal antibodies against substrate specific loop region of Plasmodium falciparum lactate dehydrogenase[J]. Immunological Investigations, 2014,43(6):556-571.
[19] Liu BH, Chu KC, Yu FY. Novel monoclonal antibody-based sensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for detecting aflatoxin M1 in milk[J]. Food Control, 2016,66:1-7.
[20] Marcossilva L, Narimatsu Y, Halim A, et al. Characterization of binding epitopes of CA125 monoclonal antibodies[J]. Journal of Proteome Research, 2014,13(7):3349-3359.
[21] Kim SH, Kim YN, Truong TT, et al. Development of a monoclonal antibody specific to envelope domain III with broad-spectrum detection of all four dengue virus serotypes[J]. Biochemical & Biophysical Research Communications, 2016,473(4):894.