IL-1018-57-PE40高效表达、纯化及细胞活性之研究

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。

High Level Expression, Purification and Cytotoxicity of IL-1018-57-PE40

The objective of the experiment is to explore the purification and production of immunotoxin .The chimeric toxin,which is composed of 40 peptides of interleukin 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 prokaryotic expression vector containing the chimeric toxin IL-10_ 18-57-PE40 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-PE40.With the application of salting out of (NH_4)_2SO_4, 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.