含绿脓杆菌外毒素A受体结合区重组蛋白的纯化  

Purification of Recombinant Protein Containing ReceptorBinding Domain of Exotoxin A of P aeruginosa

作　　者：刘晓明[1] 郭学军[1] 马从林 朱平[1] 孟锐奇[1] 李吉平 

机构地区：[1]解放军农牧大学军事兽医研究所

出　　处：《中国兽医学报》1998年第1期38-41,共4页Chinese Journal of Veterinary Science

基　　金：全军医药卫生科研基金

摘　　要：将表达含绿脓杆菌外毒素（ＰＥＡ）受体结合区基因的质粒ｐＥＴ－ＥＡＢ转化到大肠杆菌ＢＬ２１（ＤＥ３）中，经ＩＰＴＧ诱导表达了含ＰＥＡ受体结合区的重组蛋白（称ＰＥ３４）。ＰＥ３４主要以包涵体形式存在于大肠杆菌中，用溶菌酶－脱氧胆酸钠法结合超声波裂解法破碎细菌，离心制备包涵体。用２ｍｏｌ／Ｌ脲洗涤包涵体后，包涵体纯度可达７５％，在８ｍｏｌ／Ｌ脲存在条件下，ＳｅｐｈａｃｒｙｌＳ－２００凝胶滤过，ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＦａｓｔＦｌｏｗ离子交换层析纯化，获得ＳＤＳ－ＰＡＧＥ１条带的ＰＥ３４，纯度达９５．８％，得率为２４．５％。Expressing plasmid containing receptorbinding domain of exotoxin A ofP aeruginosa (PEA), pETEAB, was transformed into E coli BL21(DE3). By inducing of IPTG, a recombinant protein containing receptorbinding domain of PEA, named PE34, was expressed. PE34 formed inclusion body in expressed E coli. The expressed E coli was splitted by lysozymedeoxycholic acid sodium and supersonic. The inclusion body was prepared by centrifugation and washing with 2 mol/L urea with the purification rate of the inclusion body being above 75%. Then it was dissolved by 8 mol/L urea and further purified by Sephacryl S200 gel filter and DEAESepharose F F ionexchange chromatography. the purified PE34 appeared a single band in SDSPAGE gel with its purification rate and recovery rate being 95.8% and 24.5%.

关 键 词：绿脓杆菌外毒素A 大肠杆菌 包涵体 纯化 

分 类 号：S852.612[农业科学—基础兽医学] Q78[农业科学—兽医学]