Design Of A ProDer F1Vaccine Deliveried By The MHC Class â…¡ Pathway Of Antigen Presentation And Analysis Of Its Effectiveness For Specific Immunotherapy

Objective: To construct recombinant prokaryotic vectorpET28a(+)-DCP-Ihc-ProDer f1for expressing the fused protein DCP-Ihc-ProDerf1and explore its effect as vaccine for specific immunotherapy on large-scaleexpression and purification basis.Methods:⑴Two peptide fragments were synthesized, including the DC-bindingpeptide (DCP) and the first1-110amino-acids of the invariant chain (IhC) fortargeting the fusion protein via MHC class II pathway. The fused genes,DCP-ProDer f1and DCP-Ihc-ProDer f1, were constructed, respectively, andinserted into the prokaryotic expression vector pET28a(+). Both recombinantvectors were verified by sequencing and digested with restriction enzyme,respectively;⑵The verified vectors were transferred into the competent cells of E.coli BL21(DE3) line, and IPTG was used in low dosage to induce DCP-ProDer f1and DCP-Ihc-ProDer f1expression. Further expression and purification onlarge-scale basis did not occur until optimized conditions achieved;(3) The asthmatic murine models were developed with ProDer f1sensitization asprevious protocols, and received specific immunotherapy using DCP-ProDer f1and DCP-Ihc-ProDer f1as vaccines, followed by measurement of the levels ofcytokines IFN-γ, Interleukin (IL)-4, IL-10and IL-17in the bronchoalveolarlavage fluid (BALF) and in the supernatant of splenocyte cultures (SSCS), as wellas serum antibodies of IgE, IgG1and IgG2awith ELISA and histologicexamination of the lung tissues.Results:(1)The recombinant prokaryotic expression vectors,pET28a(+)-DCP-ProDer f1and pET28a(+)-DCP-Ihc-ProDer f1weresuccessfully constructed by respective verification with sequencing and digestingwith restriction enzyme;(2) After inducing with IPTG, the recombinant proteins,DCP-ProDer f1and DCP-IhC-ProDer f1, were expressed successfully in E. colistrain BL21(DE3) on SDS-PAGE and Western blot detection basis, and the fusedprotein was also purified on a large scale;(3) After treatment of the asthmamodels on specific immunotherapy basis using DCP-ProDer f1andDCP-Ihc-ProDer f1as vaccine, detection of the pulmonary tissues showed thatthe infiltration of inflammatory cells was alleviated as compared with the asthmagroup. ELISA results demonstrated that the levels of antigen-specific IgE andIgG1in sera from each group undergone specific immunotherapy were decreasedsignificantly, yet the levels of IgG2awere higher than the asthma group (p<0.05).Although the levels of IL-4and IL-17in BALF and SSCS were lower inimmunotherapy group than asthma group (p<0.05), the IFN-γ and IL-10levelswere increased significantly (p<0.05). In addition, DCP-IhC-ProDer f1as vaccinefor specific immunotherapy had produced superior effects to DCP-ProDer f1 and ProDer f1(p<0.05), and yet the DCP-ProDer f1and ProDer f1had nosignificant difference concerning the outcomes (P>0.05).Conclusions: The recombinant prokaryotic expression vectors,pET28a(+)-DCP-ProDer f1and pET28a(+)-DCP-ProDer f1, were successfullyconstructed, and are capable of being purified on a large scale. These fusedproteins as vaccines in specific immunotherapy for the asthmatic murine modelscan effectively alleviate allergic inflammation of airway and lung in the mice.