【摘要】 目的真核表达、纯化人IgE重链恒定区2～4区(IgECε2-4)蛋白,利用表面等离子体共振技术(SPR)捕获其相互作用蛋白。方法构建3个含不同信号肽序列的IgECε2-4重组真核表达载体,分别瞬时转染HEK293FT悬浮细胞,优化选用表达量最高的重组质粒进行大量瞬转表达,镍柱亲和层析纯化。免疫荧光技术鉴定重组蛋白与KU812细胞表面IgE受体FcεRⅠ的结合,采用SPR技术捕捉人血清中与IgECε2-4相互作用的蛋白。结果含信号肽3(SP3)的重组质粒表达量最高,表达量约为6.2 mg/L;亲和纯化获得高纯度的人IgECε2-4蛋白;免疫荧光技术鉴定显示重组蛋白IgECε2-4可与KU812细胞表面受体结合。通过SPR技术捕获到人血清中39种可能与IgECε2-4结合的蛋白。结论获得纯化的重组蛋白IgECε2-4,IgECε2-4能与KU812细胞表面FcεR1α受体结合,靶标垂钓结果显示IgECε2-4能与人血清中的39种蛋白存在相互作用或有间接结合作用。更多还原

【Abstract】 Objective To construct eukaryotic expression vectors of human IgE heavy chain 2-4 region( IgECε2-4) and purify the recombinant protein,and then capture its interacted proteins by surface plasmon resonance( SPR). Methods Three recombinant eukaryotic expression vectors of IgECε2-4 containing different signal peptides were constructed and transiently transfected into HEK293 FT suspension cells separately. The recombinant plasmid with the highest-level expression was selected to express the recombinant protein in a huge amount,and then the recombinant protein was purified by Ni-NTA affinity chromatography. The interaction between high-affinity IgE receptor( FcεRⅠ) of KU812 cell surface and IgECε2-4 was identified by immunofluorescence cytochemistry. The unknown proteins that specifically interacted with IgECε2-4 were captured from human serum by SPR technique. Results The recombinant plasmid containing the signal peptide Ⅲ showed the highest expression( 6. 2 mg/L). Highly purified recombinant protein IgECε2-4 was obtained by affinity purification.Immunofluorescence cytochemistry showed that the recombinant protein IgECε2-4 could be combined with the surface receptor of KU812 cells. Thirty-nine kinds of proteins which were likely to interact with IgECε2-4 were captured from human serum by SPR. Conclusion We obtained the purified recombinant protein IgECε2-4 that could be combined with KU812 cell surface receptor. Target fishing experiment revealed that the recombinant protein IgECε2-4 was likely to interact with 39 kinds of proteins in human serum.更多还原