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.
出处
《药物生物技术》
CAS
CSCD
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)
