抗瓜类细菌性果斑病菌单克隆抗体的制备及特性鉴定  

Preparation and Characterization of Monoclonal Antibody against Acidovorax avenae subsp. citrulli in Melons

作　　者：曾海娟[1] 王广彬 郭慧琴 邱实[1] 李建武[1] 翟绪昭 丁承超 宋春美[1] 刘箐[1] 

机构地区：[1]上海理工大学医疗器械与食品学院,上海200093 [2]徐州绿健乳品饮料有限公司,江苏徐州221006 [3]上海慧耘生物科技有限公司,上海200433

出　　处：《食品科学》2015年第23期228-232,共5页Food Science

基　　金：上海市科委高校能力建设项目(13430502400);"科技创新行动计划"长三角科技联合攻关领域项目(15395810900);上海市研究生教育创新计划项目

摘　　要：以瓜类细菌性果斑病菌燕麦嗜酸菌西瓜亚种(Acidovorax avenae subsp.citrulli,Aac)野生型菌株SD01免疫BALB/c小鼠,间接酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)法筛选获得4株稳定分泌抗SD01菌株的单克隆杂交瘤细胞株6F、6D、7E、4F,腹水抗体效价分别为1∶102 400、1∶102 400、1∶25 600、1∶51 200。采用饱和硫酸铵沉淀及Protein-G亲和层析法纯化腹水,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gelelectrophoresis,SDS-PAGE)显示纯化后的单克隆抗体(m Ab)纯度较高,纯化后单克隆抗体(2 mg/m L)效价分别为1∶12 800、1∶6 400、1∶3 200、1∶6 400,亚型鉴定结果表明4种单克隆抗体均为Ig G2 a。间接ELISA结果表明,4种单克隆抗体对8种果斑病菌结合能力不同:6F可以结合6种,6D、4F可以结合8种,7E可以结合5种。与3种非Aac近源种植物病原菌交叉反应情况为:6F、4F与2种非Aac近源种植物病原菌存在交叉反应,6D、7E与3种非Aac近源种植物病原菌均无交叉反应。Four hybridoma cell lines secreting monoclonal antibodies（m Abs） against SD01, namely 6F, 6D, 7E and 4F, were generated by immunizing BALB/c mice using wild-type strain SD01 of Acidovorax avenae subsp. citrulli（Aac） as the immunogen. Ascites antibody titers were 1：102 400, 1：102 400, 1：25 600, and 1：51 200, respectively. Ascites were purified using saturated ammonium sulfate precipitation and Protein-G affinity chromatography. Sodium dodecyl sulfatepolyacrylamide gelelectrophoresis（SDS-PAGE） analysis showed high antibody purity. The titers of purified m Abs（2 mg/m L） were 1：12 800, 1：6 400, 1：3 200, and 1：6 400, respectively. The subclass of m Abs was Ig G2 a. Indirect enzymelinked immunosorbent assay（ELISA） showed that four hybridoma cell lines had different binding capacity to eight strains of Aac and three species of non-Aac plant pathogens. 6F could bind six strains of Aac, 6D and 4F could bind eight strains of Aac, 7E could bind five strains of Aac. 6F and 4F had cross-reaction with two of the three species of non-Aac plant pathogens, and 6D and 7E had no cross-reaction with all the three species of non-Aac plant pathogens.

关 键 词：细菌性果斑病菌 单克隆抗体 制备 特性鉴定 

分 类 号：TS207.4[轻工技术与工程—食品科学]