Effective Analysis Of Lep D2, The Major Group 2 Allergen From Lepidoglyphus Destructor, As A Vaccine For Specific Immunotherapy

Objective: To clone the full-length nucleotide sequence of Lep d2, the major group allergen of Lepidoglyphus destructor, construct the recombinant plamid p ET28a(+)-Lep d2. prokaryotic expression vector that contains full length c DNA sequence of Lep d2. After inducing and purifying the recombinant protein Lep d2, Lep d2 will be used as vaccine to the murine asthmatic model for specific immunotherapy, the therapitic effect will also be assessed.Methods: The full-length nucleotide sequence of Lep d2 was synthesized according to the information of Lep d2 published on Genbank and inserted into prokaryotic expression plasmid p ET28a(+) to produce recombinant plasmid p ET28a(+)-Lep d2. After comfirming with restrict endonuclease, this recombinant plasmid was transferred into E. coli strains Rossetta. After verified by colony PCR, Lep d2 was expressed with IPTG induction to select the opitimum conditions including IPTG concentration, temperature and time. A large-scale expression of Lep d2 was performed under opitimun condition, following purification and concentration. Mice BALB/c were sensitized with dust mite crude extracte. After the murine asthmatic model was immunized with Lep d2 as specific immunotherapy. The symptoms of mice were observed, as well as pathological changes of lung tissues. Enzyme linked immunosorbent assay(ELISA) was performed to measure the levels of cytokines, include IL-5, IL-13, and IFN-γ, which were collected from bronchoalveolarlavage fluid(BALF) and spleen cell supernatant, respectively, as well as serum allergen specific Ig E(s Ig E) and s Ig G2 a.Results: The recombinant plasmid was constructed successfully by verifying with the double digestion of restriction enzyme. The results of colony PCR showed that the recombinant plasmid was also transformed into Rossetta successfully. The recombinant plasmid was expressed successfully in Rossetta(DE3) on SDS-PAGE and Western blot analysis, and the result also indicated that Lep d2 was also purified and concentrated on a large scale. After specific immunotherapy using Lep d2, the allergic inflammation changes in lung in Lep d2 SIT group were significantly alleviated. The lower level of allergen-specific Ig E(s Ig E) and higher level of s Ig G2 a were obviously observed in Lep d2 SIT group, compared with that in asthma group(p < 0.01). The level of IFN-γ of BALF and SSCS in the Lep d2 SIT group significantly increased, compared with that in asthma group(p < 0.01).On the contrary, the levels of IL-5 and IL-13 in the Lep d2 SIT group were significantly lower than that in the asthma group(p < 0.01).This indicated that Lep d2 can alleviate the allergic symptoms and inflammation of airway effectively as vaccine after SIT in murine.Conclusions: Lep d2 derive from Lepidoglyphus destructor were expessed in prokaryotic expression system successfully and could be vaccine that used to alleviate the allergic symptoms and inflammation of airway in murine.