Cloning,Expression,Characterization And Allgergic Mechanism Of Profilin, Which Is A New Allergen From Dermatophagoides Farinae

Hose dust mite is a global important source of allergens that responsible for 50% allergic patients. Among them, Dermatophagoides pteronyssinus and Dermtophagoides farinae as the two major species are associated with human allergic disease. According to the current datas, it is reported that there were 15 allergens(Der f 1-3, 6-8, 10, 11, 14 to 18 and 21, 24) from Dermtophagoides farinaes. Profilin as one of the main allergens in plant pollen and fruits has been reported. However, profilin doesn’t have been reported as an allergen of Dermtophagoides farinaes at present. We regard Dermtophagoides farinaes profilin as the object in this research, with the using of molecular biology technology and combining clinical trial datas, profilin was first proved as a kind of new allergen from Dermtophagoides farinaes. Objective:(1) Try to obtain the whole genome sequences of Dermtophagoides farinaes profilin and large amounts of recombinant protein, then identify the allergenicity of profilin.(2) Establish an allergic asthma model in mice with Dermtophagoides farinaes recombinant profilin and evaluate its immunogenicity.(3) The cross reactivity between Dermtophagoides farinaes profilin with caryota powder and mango fruit profilin is tested by ELISA.(4) Based on the dendritic cell drived from bone marrow mononuclear cells in mice, the cellular pathways of Dermtophagoides farinaes profilin is explored, which is just a pilot study.Methods:(1) According to the full genetic sequence of Dermtophagoides farinaes profilin provided by the Chinese university of Hong Kong, primers were designed, and the total RNAs of Dermtophagoides farinaes were extracted. RT-PCR was used for the cloning of profilin gene. The correct sequence which is detected through gene sequencing was connected to the expression vector Pet-28 a, then after the recombinant plasmid was transferred to E.coli BL21(DE3) and induced by IPTG, expression product purified by Ni- NTA chelating chromatography, and at last we gained high purity restructuring profilin protein. Ig E- Western blotting(Ig E- WB) experiment, the Ig E- ELISA experiment and human skin prick test(SPTs) test were conducted to test the Ig E from allergic patients serum.(2) 18 BALB/c mice were randomly divided into 3 groups, group A for dust mites extract(DME), group B for recombinant protein profilin, group C for PBS which as the normal control group. Sensitization: group A mice were treated with 100μg crude extract of dust mites in 4 mg aluminum hydroxide which is served as adjuvant through subcutaneous injection. Group B mice were treated with 100μg recombinant profilin in 4 mg aluminum hydroxide. Group C mice were treated with PBS instead of antigen. Strengthen the sensitization at 3, 7 days. At 14 days, after all of the mice were drugged, group A and group B were challenged with 50μg DME and profilin respectively through the intranasal immunization. Group C mice were also treated with PBS instead of antigen. Once time a day and 7days in a row. After 24 hours of the last challenge, airway hyperresponsiveness of all the mice were tested, serum was collected. Bronchoalveolar lavage fluid was conducted and the total number of cells and inflammatory cell number were counted. Lung inflammation was observed through pathology slice which was stained by HE. Lymphocyte proliferation was tested with MTS kits. Specific Ig E, Ig G1 in serum and cytokines INF-γ,IL-4 from spleen cells were detected by ELISA experiments.(3) The recombinant profilin of Caryota pollen and mango fruit were obtained by gene clone and protein purification technology. Serums of house dust mite allergic patients, Caryota pollen allergic patients and mango fruit patients were collected from hospitals. Cross reactivity between Dermtophagoides farinaes profilin with Caryota pollen profilin or mango fruit profilin were detected by ELISA experiments. Bone marrow-derived dendritic cells(BMDC) were induced by 20ng/ml cytokine GM-CSF and 20ng/ml IL-4. Immature DCs were divided into four groups, namely profilin stimulation group, profilin and TLR4 blockers antibody co-stimulation group and the blank control group. The levels of TIM4 expression on DCs were detected by real-time fluorescent quantitative PCR(q- PCR) and Western blotting.Results: c DNA sequence of profilin was cloned which revealed 130 amino acid open frame encoding a protein with a derived molecular mass of 14.3 k Da. p ET28a-profilin expression vector was built successfully and expressed in Escherichia coli bacteria. A large amount of soluble recombinant profilin protein was obtained and purified. Clinical skin prick test shows that there are 3 cases in 14 patients show positive reaction to Dermtophagoides farinaes profilin. Western Blotting and ELISA experiments also revealed that the ability of Dermtophagoides farinaes profilin combining with profilin allergic patients serum Ig E is 3 folds compared with healthy human serum.(2)The results of allergic asthma model as follows: group A and group B show obvious pathological changes in lungs, more serious inflammation and the number of eosinophils in alveolar lavage fluid is significantly higher(P﹤0.05) than the group C mice; The levels of Ig E and Ig G1 of group B showed higher than group C, but no obvious difference was found compared with group A; The lymphocyte proliferation and cytokine levels: the lymphocyte showed proliferation significantly when stimulated by DME and profilin. The levels of Th2 cytokines IL- 4 of group B is higher than group C, however, Th1 cytokines IFN –γ secretion level is lower than group C. But the both levels show no significant difference compared with group A.(3) Dermtophagoides farinaes profilin show 36% homology with Caryota pollen profilin and mango fruit profilin. Caryota pollen profilin showed 60% Ig E reactivity with dust mites allergic patients and mango fruit profilin showed 50% Ig E reactivity. On the contrary, Caryota pollen and mango fruit allergic patients serum as first antibody, Dermtophagoides farinaes profilin showed 20% and 26,7% inhibition rate respectively. Dermtophagoides farinaes profilin can promote TIM4 level which was expressed on the Dendritic cells surface. But when co-stimulated with TLR4 block antibody and profilin, TIM4 expression level will be depressed.Conclusion: Dermtophagoides farinaes profilin was cloned and expressed using the genetic engineering technique, Then a series of assessments were conducted which indicated that it was a new discovery allergen from Dermtophagoides farinaes. These results may lay a technical foundation for the pathogenesis of allergic disease research, diagnosis and treatment, etc.