Studies On Sensitization Mechanism Of Chitinase Allergen Of Glycoside Hydrolase Family 18 From Domestic Silkworm(Bombyx Mori) Pupa

Food allergy, which has serious impact on the human health, belongs to class Ⅰ hypersensitivity related to the body’s immune response. In nowadays, more and more people suffer the serious food allergy problem owing to a variety of different food intake in daily life, and which cause more attention of public. Domestic silkworm chrysalis is an important byproduct resource in sericulture. It is often used as a food and feed ingredients across the north and south China in a long history. On the contrary, food allergy caused by consumption of domestic silkworm chrysalis in human and animals should be attached enough importance, which has brought healthy risk to consumers and restrict the silkworm utilization in food and feed. Many allergens from glycoside hydrolase family(GHF) play an important role in the process of food allergy disease. In the paper, the purpose of this study is to obtain allergens of glycoside hydrolase family from domestic silkworm chrysalis. Furthermore, the study could provide clear knowledge for better understanding the sensitization mechanism of silkworm allergens and provide certain scientific basis for prevention and control of silkworm chrysalis sensitization.1. 96 healthy female Balb/c mice(5-6 weeks old) were used to build the food allergy model of domestic silkworm chrysalis(DSC), and subcutaneous injection(with 0.2 mg crude protein of DSC and 1 mg aluminum adjuvant) and oral administration(0.5 mg crude protein of DSC) were two treatments in the experiment. Results showed that two treatments both caused the typical characters of classⅠhypersensitivity to mice, and the allergic inflammation causes the destruction of the jejunum structure and function in mice: broken, damage, loose arrangement of the intestinal villus structure. Meanwhile, mice feet in treated group were appeared obvious allergic sensitization to swell. Furthermore, antibody specific to domestic silkworm chrysalis protein were determined by Western blot with mice sera.2. DSC protein spots specific to allergic mice sera were determined with 2-DE and Western blot reactions. And finally 15 altogether allergens from DSC protein spots were identified by MOLDI-TOF/MS/MS, and which belong to 11 different proteins(C-type lectin 10 precursor, Macrophage mannose receptor 1-like, Annexin IX isoform C, 14-3-3 protein zeta, Annexin IX isoform A, Chain A of Juvenile Hormone Binding Protein, Alcohol dehydrogenase [NADP(+)]-like isoform X1, Aldose reductase-like isoform X1, chaperonin, beta-N-acetyl glucosaminidase 2 and chitinase) with allergic mice sera and 7 altogether allergens were identified from DSC protein spots which belong to 2 different proteins(chitinase and paramyosin). And Bombyx mori chitinase(Bm Chi) belonged to GHF could cause allergy reaction to both mice and human being, therefore the Bm Chi was selected for further research.3. The American house dust mites allergen Der f 18 was found to have sequence similarity with Bm Chi protein sequence after a serial of bioinformatics analysis with chitinase from different sources. The results of Bm Chi protein analysis of secondary structure, hydrophilic-hydrophobic property, molecular flexibility, antigenicity and surface accessibility showed that the optimal antigen epitope prediction located in 23-37, 66-74, 70-82, 107-120, 139-153, 192-201, 228-236, 270-279, 308-327, 402-415, 497-510, 537-555 sites of protein sequence. Considering a scattered predicting epitope sites in Bm Chi, serials of overlapping 15-mer peptides of Bm Chi were designed and synthesized to screen the action sites with mice sera immuned by recombinant Bm Chi protein, and peptides with sequence code 536-550 and 116-130 displayed high reaction activity. Amino acid ordinal substitution and amino acid subduction method were used to redesign the peptide, and the experiment results determined the optimal antigen epitope was IDADN GDILN AMN. While the P24 and P108 peptides also exist the antigen epitope in those amino acid residues.4. The specific binding characters of Bm Chi and Ig E-Fv antibody fragment were studied based on homologous modeling, rigid docking, conventional Molecular dynamics simulation(MDS), and the steered molecular dynamics(SMD). The optimal 3D conformation of Bm Chi and combining pocket position obtained by homologous modeling were used to construct a model for predicting the combining characters of Bm Chi and Ig E-Fv. And the combining stability was evaluated by ZDock score based on shape complementary, ZRank scores based on conformation of electrostatic, Van der Waals force and desolvation energy. Results showed that small highly active peptide fragments of Bm Chi have strong hydrogen bonding interaction with mice Ig E-Fv, and the Bm Chi and Ig E-Fv Compound(BIC) displayed good electrostatic surface distribution which makes it a stable combination. Finally, the combination stability and dynamic behavior of BIC were investigated by MDS and SMD methods, and the results showed that the binding energy of BIC has small fluctuation, which mean a good stability in the process of dynamic simulation. As the simulation time goingon, the gyration radius, secondary structure composition, solvent accessible surface area and mass distance of BIC keep balance which further illustrates a closely combination. The SMD results show that the dissociation of BIC is difficult. From the above experimental results, we speculated that the high combine activity of BIC is due to the stable combination of mice Ig E-Fv and the antigen epitopes of Bm Chi.