Boosting Immune Response With The Functional Segments Of The Chicken Invariant Chain As A Universal Vector

Invariant chain is the significant chaperone of MHC class II molecules, plays animportant role in the process of antigen presentation. Because of itsnon-polymorphism，the chain named Invariant chain. Studies have shown that, Ii-keyof mouse Ii can be used as immune vectors to connect epitope, enhances the immuneresponses; and other segments of Ii could promote the effect. This article is intendedto use the functional fragments of chicken Ii as immune vectors to study thepossibility of enhancing the immune response on cross species and the mechanism ofthe immune response.According to the structure of chicken Ii, that is cytoplasmic domain, TMdomain, Ii-key (four Amino acids LRMK links N fringe of CLIP segment), CLIPand AP (two Amino acids Ala-Pro links C fringe of CLIP segment), we reconstructedchimers(Ii-F2, Cyt/TM/Ii-key/F2/AP, TM/Ii-key/F2/AP, Ii-key/F2/AP, Ii-key/F2)connecting with NDV epitope F2. Using PCR and overlap extension PCR to amplifiythe fragments with the designed primers and the preserved plasmid templatepEGFP-C1-Ii and pGEX-4T-1-F306, the fragments were inserted into the prokaryoticexpression vector pGEX-4T-1and pET-32a respectively. The results of doubledigestion and sequence showed the recombined plasmids of the chimers were formedsuccessfully.The recombined plasmids were cloned to engineering bacteria respectively. Thenwe induced the bacteria by1.0mmol/L IPTG, and identified by SDS-PAGE gelelectrophoresis. Protein expression was made after the expression condition of thefusion proteins was optimized. The induced proteins were lysed by freeze-thaw andsonication. The proteins from soluble expression (His-Ii-key/F2/AP、His-Ii-key/F2、His-F2) were extracted and purified with cutting the gel slices of the developersolution KCL, proteins from inclusion bodies (His-Ii-F2、His-Cyt/TM/Ii-key/F2/AP、His-TM/Ii-key/F2/AP) needed renature by dialysis before being purified. Six groupsof proteins were obtained and the molecular weight of the purified proteinrespectively are44.7kDa,32.3kDa,29.2kDa,24.4kDa,24.0kD,23.8kDa, the resultwas consistent with the theoretical prediction.Kunming female mice (6-8weeks old) were immunized with the purifiedproteins, supplementary immunization was made3times to obtain the specificantibody, and the antibody titers were measured by ELISA respectively. Comparedwith those immunized with F2alone, the groups immunized with Ii-key/F2andIi-key/F2/AP enhanced1.5fold and2.5fold respectively, the groups(TM/Ii-key/F2/AP, Cyt/TM/Ii-key/F2/AP, Ii-F2) enhanced about3fold. All weobtained were specific polyclonal antibody.To sum up, this experiment did prokaryotic expression of fusion protein toimmunize mice and obtain the specific antibody, the antibody titers were measured byindirect ELISA, and showed it have immunologic enhancement in various degreewhich chimers immunize mouse base on the function fragments of chicken Ii, alsoprovides valuable data to explore universal vectors of immunologic enhancement indifferent species.