Expression Of The Major Group Allergn Der F5 From Dermatophagoides Farinae And Its Effectiveness On The Murine Allergic Model For Specific Immunotherapy

Objective:To express and identify the c DNA of group 5 Dermatophagoides farinae,and analyze the effectiveness for specific immunotherapy.Methods:(1)The pair of primers were designed accoding to the known sequences of Der f5 gene.The live mites were extracted for RNA preparation and Der f5 gene fragments were amplified by RT-PCR.The product was cloned into p UC57 vector and transferred into E.coli DH-5α.The target gene obtained from the recombinant plasmid p UC57-der f5 was digested by Bam HⅠand XhoⅠ,ligated to the prokaryotic expression vector p ET28 a.The recombinant vectors were verified by sequencing and digested with restriction enzyme,respectively.(2)Then the recombinant plasmid p ET28a-der f5 was transformed into E.coli BL21. IPTG was used in low dosage to induce expression.And then further expression and purification on large-scale basis did not occur until optimized conditions achieved;(3)The asthmatic murine model was developed with Pro Derf 5 sensitization as previous protocol,received specific immunotherapy using Der f5 as vaccines. The bronchoalveolar lavage fluid(BALF) and sera were both collected, splenocyte was cultured. The number of total white blood cells and eosinophils were determined. The levels of IFN-γ, IL-5 and IL-17 cytokines in the BALF and the supernatant of cultured splenocytes(SCSS) were all measured by ELISA, as well as the levels of allergen-specific Ig E, Ig G1 and Ig G2 a in the sera. The airwayinflammation and the mucus secretion were analyzed by haematoxylin and eosin(H&E) staining.Results:(1) Two recombinant plasmids,p UC57-Der f5 and p ET28a(+)-Der f5,were successfully constructed by respective verification with sequencing and digesting with restriction enzyme;the protein,Der f5,was expressed successfully in E.coli strain BL21(DE3) on SDS-PAGE and Western blot detection basis after inducing with IPTG. And the fused protein was also purified.(2)After the treatment of the asthma model on the specific immunotherapy basis using Der f5 as vaccine, Compared with OVA and asthma groups, the lung allergic inflammation changes were alleviated significantly in SIT group. The number of total white blood cells and EOSs in SIT group greatly decreased(p < 0.01). The levels of IL-5 and IL-17 in BALF and SCSS from SIT group significantly decreased, compared with those in OVA and asthma groups, as well as the level of allergen-specific Ig E and Ig G1 in sera(p < 0.01); however, the levels of IFN-γ in BALF and SCSS from SIT group and Ig G2 a in sera were significantly increased(p < 0.01).Conclusion: The prokaryotic expression vector, p ET28a(+)-Der f5, was success- fully constructed, was capable of being purified and fused. The major allergen group 5 from Dermatophagoides farinae can inhibit airway allergic inflammation. It is as a new vaccine candidate for the treatment of allergic diseases.