摘要
以IL-10的功能短肽(40肽,即IL-10第18号至57号氨基酸)为导向部分与PE40(绿脓杆菌外毒素除去受体结合区后的剩余部分)融合分别构建了IL-101857PE40的胞质和胞周质表达质粒,其中,IL101857PE40在Rosettablue(DE3)中以高效胞质可溶形式表达,在BL21(DE3)pLysS中以胞周质分泌形式表达;表达宿主菌Rosettablue(DE3)超声波破碎后,依次通过硫酸铵盐析、疏水层析、铜离子亲和层析、阴离子交换层析纯化后,得96%重组毒素纯品;细胞活性实验、细胞ELISA和荧光标记实验表明,构建的IL101857PE40符合免疫毒素的作用机理。因此,该实验为PE免疫毒素的规模制备和纯化做了一定的有益的探索。
The objective of the experiment is to explore the purification and production of immunotoxin . The chimeric toxin, which is composed of 40 peptides of intedeukin 10（from amino acids 18 to 57） fused to a mutant form of Pseudomonas extoxin （PE） devoid of its native cell recognition domain. Two kinds of pmkaryotic expression vector containing the chimeric toxin IL-10 18-57-PF40 were constructed respectively . After induction of IPTG for 3 hours, IL-10 18-57-PE40 was expressed highly in cytoplasmic fraction in Rosettablue（ DE3）, and was directed to periplasmic space as soluble form in E. coli BL21 （DE3）pLysS . Western-blotting showed that the expressed protein could react with the specific rabbit sera against LHRH-PF40. With the application of salting out of （NH4 ）2 SO4, hydrophobic interaction chromatography, Cu-affinity chromatography and anion exchange chromatography , the purity of IL-10 18-57-PE40 was about 96 % . The cytotoxicity assay , Cell-ELISA and fluorescent antibody test support the hypothesis that IL-10 18-57 based ligand-mediated cytotoxicity can serve to target cytotoxic agents in vitro.
出处
《生物工程学报》
CAS
CSCD
北大核心
2006年第1期87-93,共7页
Chinese Journal of Biotechnology
作者简介
朱平，Corresponding author. Tel ： 86-431-7836197 ; E-mail ： pengqisheng@ sina.com
