CONSTRUCTION AND EXPRESSION OF DERMATOPHAGOIDES PTERONYSSINUS GROUP 1 MAJOR ALLERGEN T CELL FUSION EPITOPE PEPTIDE VACCINE VECTOR BASED ON THE MHC II PATHWAY.

UNLABELLED: Backgound and aims: Dermatophagoides peteronyssinus is one of the important house dust mites responsible for allergic asthma that can be tentatively managed by specific immunotherapy. The present study was to construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway. METHODS: the nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET- 28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay. RESULTS: the recombinant plasmid pET-28a-TAT-IhCDer p1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing, and the expression of the recombinant protein TAT-IhC-Der p1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p1. CONCLUSION: we successfully constructed the recombinant expression vector pET-28a-TAT-IhC-Der p1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.

Antecedentes y objetivo: el Dermatophagoides peteronyssinus es uno de los principales ácaros del polvo doméstico responsables del asma alérgica que se pueden administrar provisionalmente para una inmunoterapia específica. El presente estudio busca construir un vector que codifique epítopos de células T del grupo de alérgenos principal, el Grupo 1 de Dermatophagoides pteronyssinus como una vacuna suministrada mediante la vía MHC de clase II. Métodos: se sintetizaron las secuencias de nucleótidos de los 3 genes objetivo, incluyendo TAT, IhC y el fragmento recombinante de Der p 1 encargado de codificar 3 epítopos de célula T. Después de la amplificación de los 3 fragmentos objetivo por PCR y digestión con endonucleasas de restricción correspondientes, el gen recombinante TAT-IhC-Der p 1-3T se ligó usando T4 DNA ligasa y se insertó en el vector de expresión procariota pET28a (+) para construir el plásmido recombinante pET 28a (+)-TAT-IHC-Der p 1-3T, que se confirmó por digestión con endonucleasas de restricción y secuenciación. El vector recombinante se transformó en E. coli cepa BL21 (DE3) y se indujo con IPTG, y la proteína inducida TATIHC- Der p1-3T se detectó mediante SDS-PAGE. Después de la purificación, la proteina recombinante se confirmó por análisis de inmunotransferencia (Western blot) y se probó su alergenicidad usando el ensayo de unión a IgE. Resultados: el plásmido recombinante pET-28a-TATIHCDer p1-3T se construyó con éxito, se confirmó por digestión con endonucleasas de restricción y la secuenciación y la expresión de la proteína recombinante TAT-IHCDer p1-3T fue inducida en E. coli. Purificación con éxito verificada mediante Western blot de la proteína objetivo, que mostró una capacidad de unión a IgE más fuerte que Der p1. Conclusión: hemos construido con éxito el vector de expresión recombinante pET-28a-TAT-IHC-Der p1-3T que expresa una vacuna de epítopo de células T administrada por vía MHC II con fuerte capacidad de union a IgE. Este trabajo proporciona una base para seguir estudiando la inmunoterapia específica mediante la vía MHC de clase II.

Construction and expression of Dermatophagoides pteronyssinus group 1 major allergen T cell fusion epitope peptide vaccine vector based on the MHC II pathway / Construcción y expresión de vector de vacuna de péptido epítopo de fusión de células t de alérgeno principal del grupo 1 dermatophagoides pteronyssinus basado en la vía MHC II

Backgound and aims: Dermatophagoides peteronyssinus is one of the important house dust mites responsible for allergic asthma that can be tentatively managed by specific immunotherapy. The present study was to construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway. Methods: the nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET- 28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay. Results: the recombinant plasmid pET-28a-TAT-IhCDer p1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing, and the expression of the recombinant protein TAT-IhC-Der p1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p1. Conclusion: we successfully constructed the recombinant expression vector pET-28a-TAT-IhC-Der p1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway (AU)

Antecedentes y objetivo: el Dermatophagoides peteronyssinus es uno de los principales ácaros del polvo doméstico responsables del asma alérgica que se pueden administrar provisionalmente para una inmunoterapia específica. El presente estudio busca construir un vector que codifique epítopos de células T del grupo de alérgenos principal, el Grupo 1 de Dermatophagoides pteronyssinus como una vacuna suministrada mediante la vía MHC de clase II. Métodos: se sintetizaron las secuencias de nucleótidos de los 3 genes objetivo, incluyendo TAT, IhC y el fragmento recombinante de Der p 1 encargado de codificar 3 epítopos de célula T. Después de la amplificación de los 3 fragmentos objetivo por PCR y digestión con endonucleasas de restricción correspondientes, el gen recombinante TAT-IhC-Der p 1-3T se ligó usando T4 DNA ligasa y se insertó en el vector de expresión procariota pET28a (+) para construir el plásmido recombinante pET 28a (+)-TAT-IHC-Der p 1-3T, que se confirmó por digestión con endonucleasas de restricción y secuenciación. El vector recombinante se transformó en E. coli cepa BL21 (DE3) y se indujo con IPTG, y la proteína inducida TATIHC-Der p1-3T se detectó mediante SDS-PAGE. Después de la purificación, la proteina recombinante se confirmó por análisis de inmunotransferencia (Western blot) y se probó su alergenicidad usando el ensayo de unión a IgE. Resultados: el plásmido recombinante pET-28a-TATIHCDer p1-3T se construyó con éxito, se confirmó por digestión con endonucleasas de restricción y la secuenciación y la expresión de la proteína recombinante TAT-IHCDer p1-3T fue inducida en E. coli. Purificación con éxito verificada mediante Western blot de la proteína objetivo, que mostró una capacidad de unión a IgE más fuerte que Der p1. Conclusión: hemos construido con éxito el vector de expresión recombinante pET-28a-TAT-IHC-Der p1-3T que expresa una vacuna de epítopo de células T administrada por vía MHC II con fuerte capacidad de unión a IgE. Este trabajo proporciona una base para seguir estudiando la inmunoterapia específica mediante la vía MHC de clase II (AU)

Air-conditioner filters enriching dust mites allergen.

We detected the concentration of dust mites allergen (Der f1 & Der p1) in the air of different places before and after the starting of air-conditioners in Wuhu City, Anhui, China, and to discuss the relation between the dust mites allergen in air-conditioner filters and the asthma attack. The dust samples were collected from the air-conditioner filters in dining rooms, shopping malls, hotels and households respectively. Concentrations of dust mites major group allergen 1 (Der f 1, Der p1) were detected with enzyme linked immunosorbent assay (ELISA), and the dust mite immune activities were determined by dot-ELISA. The concentration of Der f1 in dining rooms, shopping malls, hotels and households was 1.52 µg/g, 1.24 µg/g, 1.31 µg/g and 1.46 µg/g respectively, and the concentration of Der p1 in above-mentioned places was 1.23 µg/g, 1.12 µg/g, 1.16 µg/g and 1.18 µg/g respectively. The concentration of Der f1 & Der p1 in air was higher after the air-conditioners starting one hours later, and the difference was significant (P<0.05, respectively). Additionally, dot-ELISA findings revealed that the allergen extracted from the dust was capable of reacting with IgE from the sera of asthma mice allergic to dust mites. The study concludes that air-conditioner filters can enrich dust mites major group allergen, and the allergens can induce asthma. The air-conditioner filters shall be cleaned or replaced regularly to prevent or reduce accumulation of the dust mites and its allergens.

[Construction of a vector encoding T-cell epitopes of Dermatophagoides pteronyssinus major allergen group 1 as a vaccine delivered by MHC class II pathway].

OBJECTIVE: To construct a vector encoding T-cell epitopes of major allergen group 1 of Dermatophagoides pteronyssinus as a vaccine delivered by MHC class II pathway. METHODS: The nucleotide sequences of the 3 target genes were synthesized, including TAT, IhC and the recombinant fragment of Der p 1 encoding 3 T-cell epitopes. After amplification of the 3 target fragments by PCR and digestion with corresponding restriction endonucleases, the recombinant gene TAT-IhC-Der p 1-3T was ligated using T4 DNA ligase and inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET-28a(+)-TAT-IhC-Der p 1-3T, which was confirmed by digestion with restriction endonucleases and sequencing. The recombinant vector was transformed into E. coli strain BL21 (DE3) and induced with IPTG, and the induced protein TAT-IhC-Der p 1-3T was detected by SDS-PAGE. After purification, the recombinant protein was confirmed by Western blotting and its allergenicity tested using IgE-binding assay. RESULTS: The recombinant plasmid pET-28a-TAT-IhC-Der p 1-3T was successfully constructed as confirmed by restriction endonuclease digestion and sequencing and the expression of the recombinant protein TAT-IhC-Der p 1-3T was induced in E. coli. Western blotting verified successfull purification of the target protein, which showed a stronger IgE-binding ability than Der p 1. CONCLUSION: We successfully constructed a recombinant expression vector pET-28a-TAT-IhC-Der p 1-3T expressing a T-cell epitope vaccine delivered by MHC II pathway with strong IgE-binding ability, which provides a basis for further study on specific immunotherapy via MHC class II pathway.

[Analysis of sensitization effect of chimeric allergen TAT-IhC-R8 derived from major allergen group 1 genes of dust mites].

OBJECTIVE: To explore the sensitization effect of allergen TAT-IhC-R8, derived from major allergen group 1 genes of dust mites. METHODS: Forty BALB/c mice were randomly divided into 4 groups, namely PBS group, ovalbumin (OVA) group, R8 group and TAT-IhC-R8 (TIR8) group, 10 mice each group. All the mice in OVA, R8 and TIR8 groups were treated with corresponding allergens (10 µg/ml) on the 0, 7th and 14th day by intraperitoneal injection and nebulized inhalation on day 21 with the concentration of 30 min/d for 7 days. The mice in PBS group were treated with PBS. Twenty-four hours after the last challenge, all the mice were sacrificed, their bronchoalveolar lavage fluids (BALFs) and sera were collected and their spleen cells were cultured. ELISA was performed to detect the levels of IFN-γ and IL-13 in BALFs and supernatants of cultured splenocytes (SCSs) of the mice, as well as the levels of allergen-specific IgE (sIgE), IgG, and IgG2 in their sera. The number of white blood cells and eosinophils in BALF were calculated. In addition, the airway inflammation and mucus secretion were analyzed by haematoxylin and eosin (H&E) staining. RESULTS: Compared with the PBS group, the lung inflammations of mice in the OVA, R8 and TIR8 groups were observed obviously, including inflammatory infiltration, bronchial epithelial cell breakage and falling off, as well as vasculitis. The numbers of the total white blood cells and eosinophils in BALF of mice in the TIR8 group were significantly more than those in the OVA and R8 groups (all P < 0.01). The IL-13 levels in BALFs and SCSs of mice in the TIR8 group were significantly higher than those in the OVA group and R8 group (all P < 0.01). However, the level of IFN-γ of mice in the TIR8 group was lower than those in the latter 2 groups (all P < 0.01). In addition, the levels of sera sIgE and IgG of mice from the TIR8 group were significantly higher than those in the OVA group and R8 group (all P < 0.01), but the level of IgG2a of the former was significantly lower than those of the latter groups (all P < 0.01). CONCLUSIONS: TAT-IhC-R8 can effectively stimulate lung inflammations of mice, and its sensitization effect is better than R8's.

[Effect of immunotherapy with recombinant allergen group 3 from dermatophagoides farinae in asthma mice].

OBJECTIVE: To explore the effect of specific immunotherapy with major 3 group recombinant allergen rDer f 3 of Dermatophagoides farinae in murine asthma model. METHODS: Forty BALB/c mice were randomly divided into 4 groups, namely PBS group (negative control), ovalbumin(0VA) group (positive control), rDerf3 allergen sensitization group (asthma group), and rDerf3 specific immunotherapy group(SIT group). The mice in asthma group and SIT group were injected intraperitoneally with purified rDer f 3 protein on days 0, 7 and 14, respectively, and rDer f 3 solution was inhalated from day 21 for 7 days. During the 25th-27th day, mice in SIT group were injected subcutaneously with 100 jg rDer f 3 allergen for 30 min before nasal inhalation. Mice in groups of PBS and OVA were treated with PBS and OVA, respectively. Twenty-four hours after the final challenge, all mice were sacrificed, the bronchoalveolar lavage fluid (BALF) was collected and the total number of white blood cells and the number of eosinophils were recorded. The levels of IL-5 and IFN-gamma in BALF and supernatant of cultured splenocytes were detected by ELISA, as well as the serum levels of specific IgE and IgG2, antibodies. Lung tissues were stained with haematoxylin and eosin for histological analysis. RESULTS: Compared with the asthma group, the rDer f 3-induced lung inflammation was significantly alleviated in SIT group. The total number of white blood cells [(7.03 +/- 1.38) x 10(8)/ml] in SIT group was considerably lower than that of OVA group [(22.11 +/- 3.70) x 10(8)/ml] and asthma group [(22.75 +/- 3.24) x 10(8)/ml] (P<0.01). The change trend of eosinophil leukocytes was similar with that of white blood cells. IL-5 levels in BALF [(108.20 +/- 11.02) pg/ml] and splenocyte culture supernatant [(98.34 +/- 13.06) pg/ml] in SIT group were significantly lower than that of OVA group [(182.04 +/- 13.94) pg/ml, (208.26 +/- 10.63) pg/ml] and asthma group [(195.33 +/- 15.33) pg/mL, (179.54 +/- 13.65) pg/ml] (P<0.01). Whereas, the level of IFN-gamma in BALF [(107.98 +/- 12.64) pg/ml] and supernatant of cultured splenocytes [(105.51 +/- 1.62) pg/ml] in SIT group was significantly higher than those of OVA group and asthma group (P<0.01). Compared with OVA group [(26.87 +/- 4.30) IU/ml] and asthma group [(35.25 +/- 8.84) IU/ml], a lower level of allergen-specific IgE [(9.12 +/- 3.78) IU/ml] and higher level of allergen-specific IgG2, [(38.52 +/- 6.33) microg/ml] were observed in SIT group (P<0.01). CONCLUSION: rDer f 3 allergen can reverse allergen-induced airway and lung inflammation in murine asthma model.

[Prediction and identification of T-cell epitopes in major group 3 allergen derived from Dermatophagoides farina].

OBJECTIVE: To predict and identify T cell epitopes of major group 3 allergen derived from Dermatophagoidesfarina (Der f 3). METHODS: The T cell epitopes of Der f 3 were analyzed through the sequence analysis by using the bioinformatics online tools. The five predicted peptides of T-cell epitopes were artificially synthesized. The spleen lymphocytes were co-cultured with the five T cell epitopes by using the modified MTT method and the levels of IL-2, IFN-γ, IL-4 and IL-5 in the supernatant of the cultures were detected by ELISA. RESULTS: Five T cell epitopes of Der f 3 were predicted and three of which could promote the proliferation of the mouse spleen lymphocytes. The secretions of IL-2 and IFN-γ were significantly induced and the secretions of IL-4 and IL-5 were significantly decreased by three of five prediction epitopes of Der f 3: 37GDCPYQISLQSSSHFCGG54, 98IYQHENYDSMTIDNDVALIKLKTPMT123 and 164SELQRVDIDVVSREQCDQLYS184. CONCLUSION: Three T cell epitopes of Der f 3 have been initially identified, which lays the foundation of the diagnosis and treatment of asthma.

[Prediction and identification of linear B-cell epitopes in major group 3 allergen derived from Dermatophagoides farina].

OBJECTIVE: To predict and identify the linear B-cell epitopes in the major group 3 allergen derived from Dermatophagoides farina (Der f 3). METHODS: The linear B-cell epitopes of Der f 3 allergen were analyzed based on the physicochemical properties of amino acids including antigenicity, surface accessibility, flexibility, hydrophilicity, beta-turn by online bioinformatics softwares. The eight predicted peptides of linear B-cell epitopes were artificially synthesized and incubated with three aller gic serum pools (4 serum samples in each), which were consisted of total 12 serum samples from the allergic individuals, and the strong positive epitopes were selected. RESULTS: Eight B-cell epitopes from Der f 3 were predicted successfully. Five of eight B-cell epitopes were identified with strong IgE-binding abilities followed by specific IgE assay. The amino acid sequences of them were following: 33KAKAGDCP40, 86HASGGEKIQVAEIYQHENYDSMTID110, 118LKTPMTLDQTNAKPVPLPPQGSDVKVG144, 156QEGSYSLP163 and 199DVANGGVDSCQGDSGGPVVD218. CONCLUSIONS: Five linear B-cell epitopes of Der f 3 allergen have been identified successfully. This result might provide a basis of the diagnosis and treatment for asthma.

Generation of a chimeric dust mite hypoallergen using DNA shuffling for application in allergen-specific immunotherapy.

Specific immunotherapy (SIT) is the only treatment that provides long lasting relief of allergy symptoms. Unfortunately, SIT-based traditional remedies carry the risk of producing local and/or systemic side effects. To improve the safety and efficacy of SIT, it has been proposed that SIT must utilize allergens that are hypoallergenic but hyperimmunogenic. Therefore, we used DNA shuffling to generate mutant genes encoding hypoallergens with potent immunogenicity and screened them for their capacity to modify the allergic response. We tentatively shuffled the major group 1 allergen genes from house dust mite, Dermatophagoides farinae and Dermatophagoides pteronyssinus, and discovered a novel chimeric gene, termed C 1. The gene was expressed in Escherichia coli (E. coli) and the chimeric protein C 1 was purified. An animal model of asthma demonstrated that C 1 not only decreased the production of serum IgE and IgG1, and inhibited the production of IL-4 and IL-5 in the bronchoalveolar lavage fluid (BALF). C 1 also boosted the levels of IgG2a and IFN-γ, which may demonstrate a rebalance of TH1 and TH2 allergic response. Additionally, flow cytometry showed that the immunogenicity of C 1 was higher than that of ProDer f 1, but was not significantly different from that of ProDer p 1. Our findings suggest that the C 1 is hypoallergenic and yet highly immunogenic, which makes it potentially safe and effective for use in SIT of allergic asthma.

Acaroid mite allergens from the filters of air-conditioning system in China.

Accumulation of acaroid mites in the filters of air-conditioners is harmful to human health. It is important to clarify the allergen components of mites from the filters of local air-conditioning system. The present study was to detect the allergen types in the filters of air-conditioners and assesse their allergenicity by asthmatic models. Sixty aliquots of dust samples were collected from air conditioning filters in civil houses in Wuhu area. Total protein was extracted from the dust samples using PBS and quantified by Bradford method. Allergens I and II were also detected by Western blot using primary antibody (anti-Der f1/2, Der p1/Der f2/Der p2, respectively). Ten aliquots of the positive samples were randomly selected for homogenization and sensitized the mice for developing asthmatic animal models. Total serum IgE level and IFN-γ, IL-4 and IL-5 in the bronchoalveolar lavage fluid (BALF). The allergenicity of the extraction was assessed using pathological sections developed from the mouse pulmonary tissues. The concentration of extract from the 60 samples was ranged from 4.37 µg/ml to 30.76 µg/ml. After analyzing with Western blot, 31 of 60 samples were positive for 4 allergens of acaroid mites, and yet 16 were negative. The levels of total IgE from serum IL-4 and IL-5 from the BALF in the experimental group were apparently higher than that of negative control and PBS group (P < 0.01), but there were no statistical difference compared to OVA group (P > 0.05). However,the IFN-γ level in BALF was lower compared with the negative control and PBS group (P < 0.05) but with the OVA group (P > 0.05). The pathological changes were evidently emerged in pulmonary tissues, which were similar to those of OVA group, compared with the PBS ground and negative controls. The air-conditioner filters in human dwellings of Wuhu area potentially contain the major group allergen 1 and 2 from D. farinae and D. pteronyssinus, which may be associated with seasonal prevalence of allergic disorders in this area.