Dissimilar Il-5 Dna Vaccine Eukaryotic Expression Vector Construction, Identification And Expression

Background: Asthma is one of the serious health problems in department of medicine and pediatrics. Its morbidity and mortality have increased since 1980s. A lot of investigations have been done, but the pathogenesis of asthma still remained unsolved. In present research. The imbalance of Th1/Th2 which increase of the production of Th2-cytokines (such as IL-5) and decrease of the production of Th1-cytokines (such as IFN-r), plays important role in the pathogenesis of asthma. IL-5 play a pivotal role in regulating eosinophil function (differentiation, expansion, mobilizatio, and activation). IL-5 was self-molecule. Thus, the breaking of immune tolerance against IL-5 should be a useful approach for asthma therapy by active immunity.Objective: The current studies explore the feasibility of immunotherapy of asthma with the plasmid DNA encoding human IL-5 as a vaccine by the breaking of the immune tolerance against mouse IL-5 in a cross-reaction between the human and murine xenogeneniv homdogous IL-5 molecules.Methods: We purchased the vectors of PORF9-hIL5 and PORF9-mIL5 from Invivogen company, We used Premier Primer5.0 software to design primer, The hIL5 and mIL5 gene were amplified by polymerase chain reaction (PCR). The aim gene sequences of hIL5 and mIL5 were obtained from the products which were cut with restriction enzymes , then subcloned into eukaryvotic expression vector pcDNA 3.1(+) , the resultant recombinant plasmids (named as phIL-5 and pmIL-5 respectively) were confirmed to be correctly expressed in eukaryotic COS-1 cell. Thereafter, these DNA plasmids were used as vaccines to observe whether or not they have produced antibodies against mouse IL-5. Autoantibodies against self-IL-5 were detected by Western blot.Results: The pcDNA 3.1(+)/hIL-5 and pcDNA 3.1(+)/mIL-5 have been successfully constructed through restriction enzyme analysis and sequencing. Human IL-5 and mouse IL-5 protein expression on COS-1 cells were detected by Western blot. Murine IL-5 specific autoantibodies in sera of mice with which were immunized phIL-5 could be found in Western blot.Conclusion: These observations may provide a vaccine strategy for asthma immunotherapy through the induction of the autoimmunity against the IL-5 in a cross-reaction with xenogeneichomologous gene.