| Invariant chain in antigen presented cells mainly takes part in the assembling of MHC II molecule and antigen peptide compound. Ii-Key, a portion of the invariant chain, plays a major role in antigen presentation. Recently, Ii-Key as an important immune carrier, has been proved to be able to carry single antigen or antigen peptide, stimulus IFN-y to secrete and finally greatly increase cell immunity and humoral immunity. PEP-1 is a kind of artificially-designed CPPs with the transmembrane conductance ability. It can overcome cell membrane barrier and transmembranely transduce proteins and peptides into cytoplasm and nucleus by the way of non-energy and non-receptor dependencies. In this study, we linked heterologous epitope peptides to Ii-Key and/or PEP-1 and constructed a hybrid vaccine for providing reference for Infectious Bursal Disease virus and Newcastle disease virus.Firstly, a hybrid of Ii-Key and VP2 peptide epitope(aa 197-209) of chicken Infectious Bursal Disease and HN Peptide epitope(aa345-353) of Newcastle disease virus was designed and inserted into pET-32a vector, transform E.coli Rosetta. The recombinant expression plasmids were induced with IPTG. Fusion proteins were puried by Ni SepharoseTM 6 Fast Flow, and then immuned SPF BAL b/c mice, After four times of immunization the antibody titers were detected with indirect ELISA. The results showed that valence of antibody of Ii-Key/VP2/HN hybrid vaccine treatment group was increased by 13.7 times averagely, compared with VP2/HN control group.Secondly, PEP-1 and PEP-1-Ii-Key were linked to VP2/HN peptide, respectively. The chimeras of PEP-1/VP2/HN and PEP-1-Ii-Key/VP2/HN were construced with pET-32a vector and expressed in E.coli Rosetta. The fusion proteins purified and then immuned in mice. Antibody titer were deteced by indirect ELISA. As the result shows, compared with VP2/HN control group, PEP-1 or PEP-1-Ii-Key could developed significant increases in the titer of antibody titer by 8.6 and 6.7 times, respectively (p<0.05). However, antibody titer of PEP-1/Ii-Key/VP2/HN group was obvious lower than Ii-Key carrier group(p<0.05). It indicated that PEP-1 could negatively regulate Ii-Key during the immune response.In order to investigate the mechanism underlying of the boost immune of Ii-Key and PEP-1 weather concerned with MHC Ⅱ molecule or not, we constructed the eukaryotic recombinant plasmids using the vector pmCherry-C1. Four eukaryotic recombinant plasmids, including VP2/HN, Ii-Key/VP2/HN, PEP-1/VP2/HN and PEP-1/Ii-Key/VP2/HN were transduced to E. coli strain DH5α and then co-transfected 293 T cell with Laboratory saved pEGFP-N1-MHC II a by LipofectamineTM 2000. following 24h transfection, the location situation was imaged with a laser scanning Olympus confocal microscopeunder. As the result shows, there are no apparent difference of location in cells among the experimental groups.In summary, we sucessfully constructed the hybrid peptides of VP2/HN and Ii-Key or PEP-1. Our study showed that the carrier Ii-Key or PEP-1 could carry antigen epitopes from heterologous virus and increase immune response. However, C-terminal region of PEP-1 linked to Ii-Key would reduce the humorous immune effect of Ii-Key, the mechanism underlying needs further investigation. The results provide reference for designing new vaccine based on Ii-Key of Invariant chain. |
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