Study On Specific Immunotherapy Effect Of Recombinant Blo T 21 T Vaccine

Objective:To analysis the effect of Specific Immunotherapy using reconbinant proten Blo t 21 T in asthmatic mice sensitized by the extract of Blomia tropicalis.Aad to provide a new approach to therapy asthma caused by Blomia tropicalis.Methods:?1?The three T cell epitopes?35-60AA,65-90AA,98-127AA?in gene Blo t21 T were initially synthesized using molecular biology technology.Then the three T cell epitopes were constructed as a reconbinant gene,named as Blo t 21 T.?2?Then the gene fragments were inserted into Escherichia coli expressed vector pET28a?+?to develop recombinant vector pET28a?+?-Blo t 21 T via parokaryotic expression.All recombinant vectorswere verifiedby restriction enzyme digestion and sequencing.The constructed recombinant plasmid pET28a?+?-Blo t 21 T were transferredinto the Escherichia coli expression strain E.coli BL21?DE3?.Reconbinant proten Blo t 21 T was induced in low dosage in parokaryotic vectors with different concentration gradient?0.2,0.4,0.6,0.8,1mmol/L?.And choseing the optimizing conditions that expressed Reconbinant proten to induced proten in large scale.Reconbinant proten Blo t 21 T were purified and to therapy asthma.?3?The asthmatic murine models were established by using the crude extracts of Blomia tropicalis allergen.Thirty 8-week-old female mice were randomly divided into 3 groups:A control group,B asthma group,and C immunotherapy group.Asthmatic group and immunotherapy group were injected with extracted allergen,followed by atomization challenge.In the last 3 days of immunotherapy group,specific immunotherapy for recombinant gene Blo t 21 T vaccine was given before atomization challenge.After the last atomization challenge,mouse eyeball blood,bronchoalveolar lavage fluid,and lung tissue were taken.The concentration of IgE and IgG_2aa antibodies in serum and the levels of IFN-?,IL-4 and IL-13 cytokines in BALF were detected by ELISA kit.Counting of eosinophils in bronchoalveolar lavage fluid under microscope.sections lung pathological and observing the pathological manifestations of lung inflammation.?4?Statistical Analysis:All statistical analyses in this article use statistical analysis software spss 18.0.The ANOVA?LSD-t?statistical methods were used to analyze the statistical significance of the immunotherapy group and the asthma group and the control group,respectively.When P<0.05,it was statistically significant.Results:?1?Restriction endonuclease analysis results showed that the recombinant gene Blo t 21 T was successfully synthesized and the number of base pairs was consistent with the expected.?2?SDS-PAGE electrophoresis showed that when the concentration of IPTG was 0.6 mmol/L,the target protein had a clear electrophoresis band.?3?After the purified target protein was used to treat asthmatic mice,the results of the ELISA kit showed that the serum IgE concentration was significantly lower in the immunotherapy group than that in the asthma group?P<0.05?.Serum IgG_2aa antibody levels were significantly higher in the immunotherapy group than in the asthma group?P<0.05?.The levels of IgE and IgG_2aa in the immunotherapy group were slightly higher than those in the control group,and there werestatistically significant?P<0.05?.Levels of IL-4 and IL-13 in BALF were lower in the immunotherapy group than in the asthma group(P<0.05.The immunotherapy group had higher IFN-?concentrations than the asthma group?P<0.05?.IL-4 and IL-13levels in the immunotherapy group were higher than those in the control group,and there were statistically significant?P<0.05?.IFN-?levels in the immunotherapy group were lower in the toe control group and there were not statistically significant?P<0.05?.Microscopic observation of pathological sections of the lung showed that after treatment with the recombinant gene Blo t 21 T vaccine,the degree of inflammation in the immunotherapy group was reduced,symptoms were improved,inflammatory cell infiltration was relieved,and the thickened bronchial wall was thinned.Degree of inflammation:asthma group>immunotherapy group>control group.Conclusions:In summary,we have successfully synthesized the recombinant gene Blo t 21 T,and the gene has been expressed and purified on a large scale in an expression vector.The expression product of the recombinant gene Blo t 21 T,effective relief of the vaccine,improved the symptoms of asthma in mice.