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11 024 000、 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, 1C1, 1B3 and 9D7, 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 1C1, 1B3 and 9D7 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 1C1 nor 9D7 could bind to RS-5, and 1B3 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.
Journal of Microbiology
monoclonal antibodies (mAbs)