抗STa多克隆抗体的制备、鉴定和纯化  

Preparation,Identification and Purification of Polyclonal Antibody against STa

作　　者：胡文丽[1] 匡雪 简宗辉 王皓然[1] 段锐锐 陈培富[1] 

机构地区：[1]云南农业大学动物科学技术学院,昆明650201 [2]云南省曲靖农业学校,曲靖655000

出　　处：《中国畜牧兽医》2015年第5期1082-1087,共6页China Animal Husbandry & Veterinary Medicine

基　　金：云南省高端科技人才引进计划项目(2009CI125)

摘　　要：产肠毒素大肠杆菌(ETEC)可引起人及动物急性腹泻,其关键毒力因子为耐热肠毒素(ST)。由于ST是一种具有强烈毒性但缺乏免疫原性的小分子肽,如何在保持其免疫原性的同时减弱其毒性已成为制备ST类毒素疫苗的研究热点。本试验以利凡诺法提纯兔血清白蛋白(RSA),经0.2%戊二醛活化,再与人工合成的甲醇可溶性STa偶联,十二烷基硫酸钠—聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示获得分子质量约108.5ku的完全抗原。用该偶联物免疫新西兰白兔4次,采集抗血清并用Protein A/G琼脂糖小珠进行纯化,斑点酶联免疫吸附试验(dot-ELISA)和免疫印迹(Western blotting)结果显示抗血清效价均为1∶400。试验结果表明抗STa多克隆抗体成功制备,这为筛选ST结构类似物做有益的铺垫。Enterotoxic Escherichia coli （ETEC） can cause acute diarrhea in human and animals, and its key virulent factor is heat-stable enterotoxin （ST）. ST is a peptide with small molecular weight and great toxicity but without immunogenicity,so how to keep its immunogenicity and re- duce its toxicity has become a hot research topic for preparing ST toxoid vaccine. In this study, rabbit serum albumin （RSA） was purified by rivanol method and activated in 0.2% glutaralde- hyde solution before RSA was conjugated to synthetic methanol-soluble STa, SDS-PAGE result showed that an approximate 108.5 ku complete antigen was obtained. New Zealand White rabbits were immunized with the conjugate four times,the serum was then collected and used to purify immunoglobulin by immunoprecipitation with Protein A/G Plus-Agarose. Dot-ELISA and West ern blotting result showed that the titer of the antiserum both reached 1 ： 400. These results indi- cated that anti-STa polyclonal antibody was successfully prepared, which provided the way for screening of ST structure analogues.

关 键 词：耐热肠毒素(ST) 偶联物 多克隆抗体 

分 类 号：Q813.2[生物学—生物工程]