Construction Of HuCD100-Fcε And Its Effects On Mast Cells

The incidence and severity of allergic diseases have been increasing greatly during thepast decade, partially due to the rapid industrialization, urbanization, aggravating pollutionand other reasons. According to World Allergy Organization (WAO), there are400million patients with allergic rhinitis and300million patients with asthma on our planet,with50%among them living in developing countries and lacking effective treatment.World Health Organization (WHO) has emphasized the importance of prevention andtreatment of allergic diseases for years. However, although great efforts have been madeto conquer allergy, the treatment for allergic diseases is still very difficult.The exact mechanism of allergic diseases is unclear; however, mast cells (MC) arethought to be the main effector cells of allergic reaction：allergen-specific IgE binding toIgE Fc receptor (FcεRI) on surface of MC are cross-linked by allergen，and MCdegranulation and histamine releasion will be induced, leading to the occurrence ofallergic symptom. Thus, targeted inhibiting of MC function or deleting of MC would bean effective strategy for the treatment of allergic diseases. In recent years, a variety ofinhibitory molecules on MC and their mechanisms were defined, which may provide afoundation for the design of novel targeting inhibition. Recently, CD72was foundexpressing on the surface of MC, inhibiting the function of MC when bond by its ligand,CD100. In human MC, CD72negatively regulate the Scf/Kit signaling, therefore inhibitMC proliferation, chemotaxis and production of cytokines. In the present study, we plan to construct a HuCD100-Fcε fusion protein, to targetMC as a new treatment strategy for allergic diseases: the Fcε fragment of HuCD100-Fcεwould bind the FcεRI of MC with ultra-high affinity, and the HuCD100fragment wouldbind the CD72of MC, inhibiting the Scf/kit pathway activation, MC proliferation,chemotaxis and cytokine production. The human CD100cDNA was connected withhuman IgE CH2-CH4, and the construct will then be transfected into CHO cells foreukaryotic expression of HuCD100-Fcε fusion protein. The binding activity and functionanalysis of HuCD100-Fcεs tested on human mast cells line LAD2which express bothFcεRI and CD72.Objectives:1. Production of HuCD100-Fcε fusion protein.2. Preliminarily analysis of the function of HuCD100-Fcε fusion proteins on humanmast cells LAD2.Contents and Methods:1. Construction of HuCD100-Fcε expression vector. Human Fcε gene was amplifiedby PCR, and then was inserted into HuCD100/pcDNA3.1plasmid containing humanCD100gen. The eukaryotic expression construct was termed pcDNA3.1-HuCD100-Fcε.2. Transient expression and characterization of HuCD100-Fcε fusion protein.pcDNA3.1-HuCD100-Fcε was transfected into CHO cells by Lipofectin, and theexpression of HuCD100-Fcε was analyzed by ELISA and Western blot.3. Stable transfection and characterization of HuCD100-Fcε fusion protein. CHOcell line stablely expressing pcDNA3.1-HuCD100-Fcε was selected by G418for severalweeks, and then subjected to limited dilution for single clone selection.4. Effect of HuCD100-Fcε fusion protein on MC: The effects of HuCD100-Fcεfusion protein on proliferation and histamine release of LAD2cells were analyzed in cellculture.Results:1. The eukaryotic expression construct pcDNA3.1-HuCD100-Fcε was successfullyconstructed. After transient transfection, CHO cells secreted HuCD100-Fcε fusion proteinin culture supernatant as revealed by ELISA and Western blot.2. Cell lines stably expressing HuCD100-Fcε fusion protein were successfullyobtained. Several liters of culture supernatant of stablely transfected CHO cells wereraised. 3. HuCD100-Fcε fusion protein inhibited the proliferation and degranulation ofLAD2cells.ConclusionWe proposed a novel strategy for the treatment of allergic disease by constructing afusion protein HuCD100-Fcε, and obtained the HuCD100-Fcε with good binding capacitywhich could inhibit the proliferation and degranulation of human mast cell line LAD2.Our study provided a new therapeutic approach for the treatment of urticaria and otherallergic diseases, paved the road for the future possible clinical applications.