| Objective:To investigate the mechanisms of PI3K/AKT signaling pathway in the specific immunotherapy using FABP vaccine.Methods:1)Molecular biology method was used to synthesize Blo T gene fragments that were inserted into the prokaryotic expression vector pET28a(+),construction of the recombinant prokaryotic plasmid pET28a(+)-Blo t containing pET28a(+)and Blo T with T4 DNA ligase.Double restriction endonuclease digestion and sequencing were performed to verify the recombinant plasmid pET28a(+)-Blo t,which was preservedin our laboratory;2)The recombinant plasmid pET28a(+)-Blo t was transformed into E.coli DH5a competent cells that were subjected to strain screening PCR as well as positive cloning and sequencing.After sequencing verification,IPTG was used in small dosage to induce FABP expression.Following expression conditions were optimized,target FABP protein was induced to express in large scale and purified,followed by SDS-PAGE confirmation and Western bloting detection.Finally,the FABP concentration was determined using non-interference proting assay kit(SK3071);3)Preparation of asthma mouse model with crude extract from Blomia troplicalis,forty female rats were randomly divided into four groups,namely:negative control group,asthma group,FABP immunotherapy group and PI3K inhibitor group.Mice in the asthma group,FABP immunotherapy group and PI3K inhibitor group were sensitized by intraperitoneal injection of crude extract from Blomia troplicalis at day 0,7 and 14,respectively,and challenged halation of teh allergen from the 21st day,30 min once a day for consecutive 7 days.Mice in the negative control group were treated with PBS as the protocol described above.By day 25,26 and 27,mice in the FABP immune therapy group and PI3K inhibitor group were undegone treatment with FABP vaccine by intraperitoneal injection half an hour before inhalation,wherease those in PI3K inhibitor group were treated with PI3K(LY294002)inhalation 1.5h before FABP administration,and mice in the negative control group were managed with PBS by both intraperitional injection and inhalation using similar dosage.Mice in the three groups except negative controls were further treated by inhalation of solvent DMSO in the same dosage before immunotherapy;4)Samples were obtained in mice in individual group within 24 h following final inhalation excitation for examination.ELISA was used to measure the levels of IL-4,IL-5,IL-13,IL-17,IL-10 and IFN-y in the supernatant from BALF and spleen cells as well as content of serum specific antibodies IgE,IgGi and IgG2a.Pulmonary tissue sections were prepared,and cells in the BALF were categorized and counted.Finally,the protein was extracted from the lung tissues for detecting the signaling pathway and phosphorylation,including Akt,p-Akt,I?B-a and p-I?B-a.Results:1)Double enzyme digestion and sequencing indicated that the recombinant plasmid pET28a(+)-Blo t was successfully constructed;2)SDS-PAGE and Western bloting confirmation showed that the target FABP protein was successfully induced and purified in our experiment,and the purified FABP concentration was 1.12 mg/ml,measured by teh standard curve;3)ELISA findings indicated evident decrease of IL-4,IL-5,IL-13 and IL-17 levels in the culture supernatant of BALF,significant increase of IL-10 and IFN-?,markedly fall of serum specific IgE and Ig Gi contents,yet increased IgG2a in spleen culture from mice of FABP immune therapy group.The difference was significant among indicators(P<0.01).Significantly elevated IL-4,IL-5,IL-13 and IL-17 levels,yet decreased IL-10 and IFN-y levels,evident increase of serum specific IgE and IgG contents,and decreased IgG2a in the culture supernatant of BALF in mice treated with PI3K as compared with the indicators observed in mice in teh FABP immunotherapy group.The difference was significant among indicators(P<0.01).However,the above indicators remained no significant difference between the asthma group and PI3K inhibitor group(P>0.05).Examination of the pathological sections of lung tissues showed well improved pulmonary inflammation,mitigated infiltration of the inflammatory cells to a certain degree,better maintained configuration of the epithelia at airways and significantly alleviated asthma symptoms in mice received FABP immunotherapy.By comarison with teh FABP group,mice in the PI3K inhibition group exhibited visually widened alveoli,thickened airway walls,increased intramural inflammatory cells and secretions in the airways.These symptoms were indentical with asthma.Cell categorizing and count in the BALF showed that the total cell and eosinophil counts were significantly decreased in mice treated with FABP.The difference was significant compared to the indicators of mice in asthma group(P<0.01),yet was not significant compared to the indicators of mice treated with PI3K inhibition(P>0.05).Western bloting detection of the signaling and phosphorylation(Akt,p-Akt,I?B-a and p-I?B-a)in proteins of lung tissues demonstrated that mice in the FABP immune therapy group and PI3K inhibitor group had significantly decreased mean values of p-Akt/Akt and p-I?B-a/?-actin,yet elevated I?B-a/?-actin level.All indicators were significantly different(P<0.01).Increased p-Akt/Akt,p-I?B-a/?-actin,and decreased FABP group,with the indicators being different(P<0.01),however,the above indicators remained no significant difference by comparison with PI3K inhibition group and the asthma group(P>0.05).Conclusion:The recombinant plasmid of pET-28a(+)-Blo t was successfully constructed,and the target FABP protein was successfully expressed and purified in large scale in our experiment;Specific immunotherapy with the recombinant FABP for asthmatic mice sensitized by the crude extract prepared from Blomia tropicalis can improve the asthma symptoms to a certain extent;Molecular mechanisms of the recombinant FABP protein as a vaccine for immunotherapy of asthma mice may be achieved by regulating the signaling pathway of PI3K/AKT/NF-?B.To provide new ideas for the follow-un treatment of asthma caused by mites. |
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