IL-10_(23-57)-PE40可溶表达、纯化及其细胞活性

以IL-10的功能短肽(35肽,即IL10第23至57氨基酸残基)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)融合分别置入pet20b(+)和pet28a(+)构建重组毒素IL102357PE40的两种表达质粒,其中置于pet20b(+)的重组毒素在BL21(DE3)pLysS中以周质分泌可溶形式表达,置于pet28a(+)的重组毒素在Rosettablue(DE3)中以高效胞质可溶形式表达;依次通过硫酸铵盐析、疏水层析、阴离子交换层析、铜离子亲和层析纯化周质分泌成份,得90%重组毒素纯品;细胞活性实验表明,该重组毒素只对单核巨噬细胞有杀伤作用;细胞ELISA显示,该重组毒素对单核巨噬细胞的杀伤作用(IC50为13.9pmolL)符合绿脓杆菌外毒素的作用机理.

Soluble Expression,Purification and Cytotoxicity of IL-10_(23-57)-PE40

An immunotoxin functions by binding to the target cell,becoming internalized,and exerting a toxin action which kills the cell.To develop chimeric protein in which the targeting and toxic portions are combined into a single molecule using recombinant DNA technology,this immunotoxin can target immunocyte populations to prevent organ graft rejection and ameliorate symptoms of autoimmune diseases.The chimeric protein composed of 35 amino acids of interleukin 10(IL-10_ 23-57)fused to truncated form of pseudomonas extoxin(PE) devoid of its native recognition domain,two types of proeukaryonic expression vector,pet-20b-pet-IL-10_ 23-57-PE40 and pet-20a-IL-10_ 23-57-PE40,were constructed, following transfecting into E.coli BL21(DE3)pLys.IL-10_ 23-57-PE40 was expressed in soluble periplasmic and cytoplasmic fraction,respectively. SDS-PAGE showed a single band 43 kD as expected.And Western blot revealed that the expressed protein could specifically bind with anti-IL-10-PE40 antibody.The immunotoxin was further purified to 90% purity with (NH_4)_2SO_4 precipitation,Octy1 Sepharose 4FF,DEAE-Sepharose 4 FF and Cu-based affinity chromatography consecutively.Cytotoxicity assay demonstrated that treatment of mononuclear macrophage carcinoma cell line,RAW264.7,IL-10_ 23-57-PE40 resulted in the cell death with highest efficacy(IC_ 50:13.9 pmol/L),while for bone marrow carcinoma cell line SP2/0 with less efficacy.These results suggest that recombinant immunotoxin IL-10_ 23-57-PE40 targets and kills specific immunocytes and has the potential as therapeutic agent for autoimmune diseases.