ad52knob 蛋白在大肠杆菌中可溶性表达及其活性检测.pdf

Ad5knob 蛋白在大肠杆菌中可溶性表达及其活性检测 赵颜忠 ,陈玉祥 ,文 　路 , 刘 　霆 ,朱晒红 ( 中南大学湘雅三医院医学实验中心 湖南长沙 4 100 13) Sol uble Expression , Purif ication and Characterization of Ad5knob Protein ZHA O Yanzhong , C H EN Yuxiang , W EN L u , L IU Tin , ZHU Xiho ng ( M e d ical E x p eri ment al Center of 3 rd X i angy a H osp it al , Cent ral S out h Uni vers ity , Changs ha 4 100 13 , Chi na ) Abstract : A d5kno b gene f ro m A d Ea sy p la smind DN A wa s amp lified by p olymera se chain reactio n ( PCR) an d cloned into an E. col i exp re ssion vector p Q E30 dige st ed wit h B am H I and H i n d III. Aft er induction wit h IP T G , t he t ran sfor med E. col i st rain M 15 exp re ssed A d5knob gene effi cient ly . Moreover , t he reco mbinant A d5kno b p rot ein mainly exi st ed a s a soluble p rot ein . Aft er one st ep p urification wit h Ni2 + N TA affinit y chro mato grap hy , t he p rot ein wa s p urified to nearly ho mo geneit y . Nt er minal amino acid sequencin g in dicat ed Nt er minu s of p urified A d5knob . When F I TClabeled A d5knob t ran sf er s Hela cell s , it can sp ecially bind to t he recep tor to t he surf ace of Hela cell s and ent er t he cyto sol . It sugge st s t hat t he reco mbinant A d5knob p rot ein i dentified and p urified i s to be suit able for nonviral gene car rier s a s a t ar get p rot ein in f ut ure st u d ie s. Key words : A denovir u s ; Pro karyotic exp re ssion ; Knob p rot ein ; Det er minatio n of activit y 摘要 :采用 PCR 技术 , 从 A d Ea sy1 质粒 DN A 中获得 knob 全长序列 , 克隆入p GEMT vector 中。DN A 测序鉴定 后 , 构建 p Q E30/ knob 重组表达载体 , 转化大肠杆菌 M 15 p R EP4) , IP T G 诱导表达腺病毒 5 型纤维蛋白的 knob ( ) 功能域 A d5k