上海海洋大学学报2024，Vol.33Issue(2)：389-397,9.DOI:10.12024/jsou.20230304124
甲壳类和贝类原肌球蛋白的广谱单克隆抗体制备与鉴定
Preparation and characterization of universal monoclonal antibody specific for tropomyosin in crustacean and shellfish
摘要
Abstract
Tropomyosin(TM)is considered to be a major allergen of crustaceans and shellfish.To address the increasing risk of crustacean and shellfish allergy,in this study,TM enrichment from shrimp(Litopenaeus vannamei)was used as the immunogen of BALB/c mice.A monoclonal antibody(Mab)cell line 2F8 against TM allergen of Litopenaeus vannamei was obtained by hybridoma cell fusion techonology.The immunological properties of Mab 2F8 including titer,specificity,antibody isotype and binding region were characterized using ELISA,Western Blot and bioinformatics.Moreover,its application feasibility was discussed using a sandwich colloidal gold test strips constructed by Mab 2F8 and rabbit polyclonal antibody specific for TM.ELISA and Western Blot results showed that the antibody subtype of Mab 2F8 was IgG1,with an antibody titer of 1.28×105 and an IC50 value of 20.21,indicating a high affinity to TM.It was found that the purified Mab 2F8 had specific reaction with the TMs in crustaceans,shellfish and squid.Three potential binding regions in amino acid sequence AA0-31,AA38-43 and AA58-67 of TM were found according to bioinformatics analysis techniques.Detection results by test strips showed that 5 ng/mL TM(equivalent to 12.5 µg/kg)of white prawn shrimp could be visually detected within 10 min,showing that the obtained Mab 2F8 could be used for the detection of TM in the muscle of crustaceans and shellfish.Our study indicated that the purified 2F8 Mab had high affinity and good application value,which could be used for identification and detection of allergen TM in shrimps,crabs,shellfish and squid.This study provides important biological materials for the identification or detection of allergen TM and technical support for the development of rapid detection kit of TM in the future.关键词
原肌球蛋白/单克隆抗体/凡纳滨对虾/甲壳类/贝类Key words
tropomyosin/monoclonal antibody/Litopenaeus vannamei/crustacean/shellfish分类
轻工纺织引用本文复制引用
陆佳达,栾宏伟,闫艺方,施文正,卢瑛..甲壳类和贝类原肌球蛋白的广谱单克隆抗体制备与鉴定[J].上海海洋大学学报,2024,33(2):389-397,9.基金项目
国家自然科学基金面上项目(32172324) （32172324）
国家重点研发计划项目(2019YFD0902003) （2019YFD0902003）
