Pathogenetic Study Of The Recombinant Solenopsis Invicta Buren Venom Allergen Soli Ⅳ

The red imported fire ant,solenopsis invicta,is an aggressive predator and has been recognized as a serious public health hazard.Stings from these ants have become the leading cause of insect venom allergic reactions and death.Therefore,the objective of this study was to research the pathogenesis of solenopsis invicta buren venom allergen sol iâ…£by injecting the recombinant solenopsis invicta buren venom allergen sol iâ…£(r Sol iâ…£) to rabbits.In the present study,we use bacillus coli to express the r sol iâ…£at the first time in domestic.The r sol iâ…£was highly expressed when the engineered bacteria BL-21 were induced by 1.5 mmol IPTG at 37℃for 5 h.After ultrasonication,the r sol iâ…£was released into the supernate,verifying it to be a soluble protein.Subsequently,the r sol iâ…£was highly purified by Ni²⁺ resin chromatography column.The anaphylaxis of venom protein was researched by injecting r sol iâ…£to rabbit and mouse.Results indicated that the recombination protein could cause allergic response only in rabbit,which proved the biological activity of recombination protein and more sensibility of rabbit than mouse.About 40%of rabbits showed allergic response.Therefore,we selected anaphylactic rabbits by skin tests for subsequent immunotherapy tests.After injecting the r sol iâ…£in rabbit by means of low dose and many times,we discovered that the protein can produce a marked therapic effect.The recombination protein Sol iâ…£was used to stimulate peripheral blood lymphocyte of rabbit combined with LPS and ConA in vitro,and the lymphocyte proliferation was measured by the method of MTT assay.Combined with LPS,25μg/mL,50μg/mL,75μg/mL of recombinant Sol iâ…£treatment could significantly(P＜0.05) promote lymphocyte proliferation of peripheral blood compared with LPS treatment alone.When combination with ConA,15μg/mL,25μg/mL,50μg/mL and 75μg/mL of recombination Sol iâ…£could promote significantly lymphocyte proliferation of peripheral blood compared with ConA alone.We also did the sensitization test,and the skin,liver and lung of experimental rabbits were detected by histopathological methods.Skin:the dermis layer necrosed,considerable eosinophile granulocyte and inflammatory cells appeared surrounding the focus of necrosis. Liver:vesicular degeneration and granular degeneration could be found in hepatocyte,lots of eosinophile granulocyte appeared in hepatic sinusoid.Lung:blood capillary in alveolar wall was engorgement,many eosinophile granulocyte appeared in alveolar wall.After 32 h of injecting with the r sol iâ…£,IL-4 and IL-10 mRNA in spleens and lymph node were significantly higher(P＜0.01) than the control,but IFN-γ,mRNA was significantly decreased(P＜0.01).The level of IL-4 mRNA in spleen was 8.3 times higher than the control group,and the level of IL-10 mRNA in spleen was 7.2 times higher than the control group,but the level of IFN-γ,mRNA was 13.4%in comparison with the control group in spleens.The IL-4 mRNA level in lymph node was 6 times higher than the control group,and the IL-10 mRNA in lymph node was 16.4 times higher than the control group, but the IFN-γmRNA level was 12.8%in comparison with the control group in lymph node. There were no significant differences of IL-6 mRNA in spleens and lymph node between the injected group and the control group.In conclusion,r sol iâ…£could significantly promote proliferations of T and B lymphocyte in vitro at a certain concentration,and hypersusceptibility typeâ… would be induced in rabbit of hypersusceptible habitus.