| Objective:To construct the transgenic recombinant vector of TRBO-Der f1and its expression in tobacco and to explore the effect of allergen specific immunotherapy of the major allergen group1(Der f1) from Dermatophagoides farinae (D. farinae) expressed in tobacco to allergic asthma.Methods1. Construction of transgenic recombinant vector of allergen group1from D. farinae and its expression in tobaccoThe total RNA was extracted from D. farinae, the Der f1gene was amplified using total RNA as template by reverse transcription polymerase chain reaction (RT-PCR) and inserted into the transient expression vector TRBO for constructing the recombinant plasmid TRBO-Der f1. The recombinant plasmid was identified using digestion and PCR after transformed to Agrobacterium tumefaciens (A. tumefaciens) line GV3101. Tobacco leaves were harvested at3,4,5,6d respectively, following infiltration with GV3101containing recombinant plasmid TRBO-Der f1, and Der f1proteins were identified by SDS-PAGE electrophoresis.2. Immunotherapy with the major allergen group1from D. farinae expressed in tobacco to allergic airway inflammation in mice100BALB/c mice were randomly divided into5groups, namely PBS (negative control), ovalbumin (positive group), prokaryotic expression Der f1(pDer f1), tobacco expression Der f1(tDer f1), tobacco expression Der f1treatment (tDer f1-T). PBS group were treated with PBS all the time, the remaining four groups were sensitized intraperitoneally with OVA, pDer f1, tDer f1, tDer f1respectively on day0,7,14, and then used inhalation method to challenge on day21for7days. Twenty-four hours after the last challenge, mice in group PBS, OVA, pDer f1, tDer f1were sacrificed and the mice in tDer f1-T were treated continuely used the major allergen group1from D. farinae expressed in tobacco. The lungs were made into slices stained by hematoxylin and eosin in order to observe the pathologic changes in lungs. The bronchoalveolar lavage fluid (BALF) was collected to determine the number of the total cells, neutrophils, lymphocytes and eosinophils by Liu’s stain. ELISA was used to assay the level of cytokines (IL-2, IL-4, IL-5, IL-17, and IFN-y) in BALF and in the supernatant of splenocyte culture (SCCS) and the level of D平【er f1-specific IgE, IgG1in the sera.Result1. Construction of transgenic recombinant vector of allergen group1from D. farinae and its expression in tobaccoElectrophoretic analysis and sequencing showed that the Der f1gene, with627bps, was cloned successfully and the recombinant plasmid TRBO-Der f1was transformed to A. tumefaciens line GV3101as expected. Der f1proteins were expressed highly in tobacco leaves at5,6d after infiltration.2. Immunotherapy with the major allergen group1from Dermatophagoides farinae expressed in tobacco to allergic airway inflammation in miceCompared with group OVA, the lung allergic inflammation changes of group tDer f1-T were significantly alleviated. The numbers of total cells and eosinophils in group tDer f1-T were greatly decreased, while the level of specific IgE and IgG1was significantly lower in group tDer f1-T than that in group OVA. The level of IL-4, IL-5, IL-17in group tDer f1-T was significantly decreased compared with that of group OVA, and the level of IL-2, IFN-y in group tDer f1-T significantly higher than that of group OVA, IL-4, IL-5, IL-17released from culture supernatants of splenocytes in group tDer f1-T was significantly decreased than those in group OVA; IL-2, IFN-y released from culture supernatants of splenocytes in group tDer f1-T was significantly increased than those in group OVA.Conclusion The transgenic vector of recombinant plasmid TRBO-Der f1was constructed, and the Der f1proteins were expressed in tobacco leaves successfully. The results in the present investigation would provide a basis for the further study on the transgenic plant vaccine. The major allergen group1from D. farinae Expressed in Tobacco can inhibit airway allergic inflammation. Figure [8] table [7] reference [75]... |
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