创伤弧菌外膜蛋白多克隆抗体的制备及其特性分析  

Preparation and characterization of polyclonal antibody against outer membrane proteins of Vibrio vulnificus

作　　者：古小莉[1] 李永贤[1] 李惠青 陈洁文[1] 李刘冬[1] 

机构地区：[1]中国水产科学研究院南海水产研究所农业部水产品加工重点实验室农业部水产品贮藏保鲜质量安全风险评估实验室,广东广州510300

出　　处：《中国渔业质量与标准》2017年第5期45-50,共6页Chinese Fishery Quality and Standards

基　　金：中国水产科学研究院南海水产研究所中央级公益性科研院所基本科研业务费专项资金资助(2015TS18;2009TS02;2015TS15);广东省水产品质量安全专项项目(GDSCZA2015009);国家农产品质量安全风险评估重大专项(GJFP201501003)

摘　　要：[目的]制备创伤弧菌特异性抗体,为建立免疫学快速检测创伤弧菌提供参考依据。[方法]以创伤弧菌外膜蛋白作为抗原,将抗原分多次免疫新西兰大白兔获得特异性多克隆抗体,采用蛋白G亲和层析法纯化多克隆抗体,通过间接ELISA和Western Blot测定创伤弧菌外膜蛋白多克隆抗体的效价、敏感性和特异性。[结果]实验获得了纯度较高的创伤弧菌外膜蛋白多克隆抗体,效价为1∶64 000;抗体对创伤弧菌具有高的特异性,它与多株致病菌均无明显交叉反应,其对创伤弧菌检测灵敏度为103CFU/m L;Western Blot分析表明,制备的多克隆抗体能识别创伤弧菌外膜蛋白,创伤弧菌外膜蛋白具较强的抗原性。[结论]创伤弧菌外膜蛋白多克隆抗体具有较高特异性和灵敏性,可应用于创伤弧菌的快速、灵敏的免疫学检测。[ Objective] This study aimed to prepare high specific antibody against Vibrio vulnificus, and provide refer- ences for rapidly screening Vibrio vulnificus by immunological method. [ Methods ] The polyclonal antibody was ob- tained by injecting the antigen, which was the outer membrane proteins of Vibrio vulnificus, to New Zealand rabbits in several times with increasing dose, and purified by protein G affinity chromatography. Indirect ELISA and Western Blot assay were conducted to characterize the titer, sensitivity and specificity of polyclonal antibody. [ Results ] The polyclonal antibody prepared had high purity, which titer was 1：64 000. Moreover, the antibody had high specificity for Vibrio vulnificus. It was no obvious cross reaction with other multiple pathogenic bacteria. The minimum threshold detection limit was 103CFU/mL in pure culture of Vibrio vulnificus. Western Blot result showed that the polyclonal an- tibody could specially identify the outer membrane proteins of Vibrio vulnificus and the outer membrane proteins should have good antigenicity. [ Conclusion ] The polyclonal antibody against outer membrane proteins of Vibrio vulnificus was high specificity and sensitivity, which could be used to rapidly screen Vibrio vulnificus.

关 键 词：创伤弧菌 外膜蛋白 多克隆抗体 WESTERN BLOT ELISA 

分 类 号：R392-33[医药卫生—免疫学]