| Objective Asthma, as one of type I allergy, was caused by IgE-mediated hypersensitivity. IgE was produced by specific B lymphocytes when allergen was first introduced to the body, then binding to IgE-specific high-affinity receptor (Fc?RI) on the surface of mast cells or basophils through its Fc-region, making these cells sensitized. In the allergic reaction, re-esposure of sensitized patients with the same allergens, leads to bind to IgE on mast cells or basophils, cross-linking of receptors, triggering the release of inflammatory mediators to cause a series of allergic clinical symptoms, such as smooth muscle contraction, enhanced gland secretion. Given the important role of IgE in asthma, scientists are always trying to neutralize circulating IgE to inhibiting the binding to Fc?RI, which was the new breakthrough in the treatment of asthma. Therefore, the study of blocking IgE binding to Fc?RI was the hotspot of asthma and allergic drugs research.Research showed that the Fc-region of IgE comprising C?3 and C?4 is sufficient for binding to Fc?RI with high affinity, similar to native IgE. Therefore, our laboratory using recombinant human IgE C3-C4 fragments instead of the native IgE to select anti-IgE antibody from human IgG genes library, which was constructed from peripheral lymphocytes of 300 healthy volunteers and the diversity was approximately 9×107. One positive clone named as lib-8 was obtained with specificity to recombinant human IgE C3-C4 fragments. This study aims to construct a murine asthma model with high level of IgE, for preclinical evaluation of the therapeutic use of anti-IgE antibody. Analysis was also conducted to confirm the binding site to IgE of lib-8. Then in vivo and in vitro tests were conducted to evaluate the effect of the lib-8.Methods 1. A murine asthma model by sensitizing and challenging 6-8 weeks C57BL/6 female mice with Dermatohpagoides farinae extracts was conduted to analog the process of dust mite-induced asthma model with high level of IgE and lay the foundation of exploring the effect of anti-IgE antibody therapy in asthma.2. RosettaAntibody homology modeling was conducted to construct and predict the molecular model of the variable region of lib-8. Antigen-antibody RosettaDock docking was used to predict the IgE C3-C4 antigen and the lib-8 antibody possible interaction epitope. The IgE C3, IgE C4, IgE C4-link gene was obtained from IgE C3-C4-pET-19b vetctor prepared before by PCR, and was ligated with the pET-19b vector, expressed in E.coli strain BL21 (DE3) star pLysS as inclusion. These recombinant proteins were purified using Protein Refolding Kit (Novagen, Madison, WI) by Glutathione redox system according to the manufacturer's instructions. ELISA and Western blotting were conducted to analyze the binding site of lib-8 to IgE.3. An in vivo test was performed through asthma murine model descriped as above, 100?g /times lib-8 treatment for 5 consecutive days, and 300?g on the sixth day to verify the ability of lib-8 to inhibit or neutralize free IgE.4. In vitro test was conducted to dadicate the ability of lib-8 to inhibit IgE receptor-mediated degranulation of mast cells sensitized with murine anti-DNP-IgE and challenged with DNP-BSA based on P815 mast cells.4-methylumbelliferyl-N-acetyl-?-D-glucosaminide was used as substrate and fluorescence measurement was performed to quantify the release of ?-hexosaminidase.Results 1. A murine asthma model was successfully conducted with high level of IgE including total IgE and the specific group 2 allergen of Dermatohpagoides farinae IgE (rDer f 2-sIgE), which makes it possible to the study of anti-IgE antibody therapy of asthma. The number of total cells and eosinophils in BALF of asthma group was significantly higher than that in the control group(P<0.01). Inflammatory cells were obviously observed in the lung tissues of the asthma group. The level of total IgE (P< 0.001) and rDer f2-sIgE (P< 0.01) in serum enhanced significantly. The level of IL-4 in BALF was significantly increased (P< 0.05), while the INF-y was decreased (P< 0.01).2. Recombinant human IgE C3, IgE C4, IgE C4-link proteins were obtained and the binding site of lib-8 to IgE was identified to be Cs3 region by ELISA and Western blotting.3. The results of in vivo test suggested the possibility of lib-8 to treat asthma. The number of total cells and eosinophils in BALF of lib-8 treatment group was significantly lower than that in the asthma group (P< 0.01). Inflammatory cells were obviously decresed in the lung tissues of the lib-8 treatment group. The level of total IgE (P< 0.01) and rDer f 2-sIgE (P< 0.05) in serum reduced significantly. The level of IL-4 in BALF was significantly decreased (P < 0.05), while the INF-y was no significant difference.4. The results of in vitro tests dedicated that lib-8 could inhibit the degranulation of P815 mast cells, the release of ?-Hexosaminidase (P< 0.05) was significantly decreased and the degranulation was fewer observed by optical microscope (X 400) after the treatment of lib-8.Conclusion A murine asthma model with high level of IgE was successfully constructed. The engineered anti-IgE antibody, lib-8, screened before in our laboratory had good reactivities, whose binding site to IgE was C?3 region. |
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