Constant Connaught Medical Technology Co.,Ltd. In Guangzhou City

Objective To construct eukaryotic expression vectors of human IgE heavy chain 2-4 region (IgECepsilon2-4) and purify the recombinant protein, and then capture its interacted proteins by surface plasmon resonance (SPR). Methods Three recombinant eukaryotic expression vectors of IgECepsilon2-4 containing different signal peptides were constructed and transiently transfected into HEK293FT 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 (FcepsilonR I) of KU812 cell surface and IgECepsilon2-4 was identified by immunofluorescence cytochemistry. The unknown proteins that specifically interacted with IgECepsilon2-4 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 IgECepsilon2-4 was obtained by affinity purification. Immunofluorescence cytochemistry showed that the recombinant protein IgECepsilon2-4 could be combined with the surface receptor of KU812 cells. Thirty-nine kinds of proteins which were likely to interact with IgECepsilon2-4 were captured from human serum by SPR. Conclusion We obtained the purified recombinant protein IgECepsilon2-4 that could be combined with KU812 cell surface receptor. Target fishing experiment revealed that the recombinant protein IgECepsilon2-4 was likely to interact with 39 kinds of proteins in human serum.