细胞毒素Gelonin的亚克隆、表达与纯化  

Subclone, Expression and Purificatin of Cytotoxin Gelonin

作　　者：李卓玉[1] 郭晨云[1] 袁静明[1] 

机构地区：[1]山西大学生物工程研究中心,山西太原030006

出　　处：《药物生物技术》2002年第4期191-195,共5页Pharmaceutical Biotechnology

基　　金：山西省自然科学基金资助项目(20011027)

摘　　要：将分别含有细胞毒素gelonin基因片断的重组子pUC-gell和pUC-gel　Ⅱ,按照预先设计的酶切位点,通过亚克隆得到含有gelonin全基因的重组子pUC-gel。然后将pUC-gel与pET28a同时用NdeI/EcoRI双酶切,相关片段构建成表达重组子pET-gel,并进行DNA序列测定。工程菌 E.coli BL21/pET-gel表达产物经两步SP-Sepha-rose柱层析,可得电泳纯的重组 gelonin,分子质量为28000u。无细胞体系蛋白质合成实验表明,其地（使蛋白质合成抑制50％所需浓度）为35μg/L。酶联免疫实验为阳性。Gelonin, a kind of ribosome inactivating proteins (RTPs), can couple with the cell-specific antibody to be an immunoconjugate which could be used as a potential therapeutic agent for cancers and some autoimmune diseases. In this paper, a recombinant pUC-gel was first constructed by cloning gelonin gene synthesized chemically to vector pUC118.Subsequently, both pUC-gel and vector pET28a were simultaneously cleaved with NdeI/EcoRI and the relevant fragments were reconstruted to be an expression recombinant pET-gel. After transformation, the engineered strain E.coli BL21/ pET-gel can express a soluble target protein in LB medium supplemented with kanamycin after IPTG induction. By two steps of chromatography on SP-Sepharose FF column, a homologous product with molecular weight of 28kD was occurred by SDS-PAGE analysis. In addition, it was also shown from the experiment of protein synthesis with cell-free system that IC50(e.g. reaching 50% inhibition) was 35 ug/L,which was corresponded to two folds of native pure gelonin assayed by reticulocyte lysate translation system. At the same time, the test of ELBA was obviously positive too.

关 键 词：细胞毒素 GELONIN 亚克隆 表达 纯化 

分 类 号：Q51[生物学—生物化学]