Preparation And Conformational Stability Analysis Of AFP/HLA-A2 Dimer Linked By Fos-jun

HLA-A2, one of classic MHC?molecules, is composed of a heavy chain(H chain or alpha chain, including ?1, ?2 and ?3 domains) and a light chain(L chain or ?2-microglobulin), wherein ?3 domain is noncovalently linked to ?2m. The antigenic peptides bind the polymorphic groove between ?1 and ?2 domains, when the antigenic peptides binding to MHC molecules can form the p MHC complex, and then displayed to CD8+ T cell receptors, which induces specific cellular immune response. Therefore, substantial connections between ?-chain, ?2m and antigen peptide are the foundation for native conformation and normal biological function of MHC?molecule. In order to obtain the integrity of p MHC, we have to ensure the stable connections among them. Some studies have reported, it is an invalid when TCR binds to p MHC monomer. Therefore the late researches improve affinity between p MHC and TCR by making the p MHC multimerization, and confirme the solube p MHC dimer can bind to TCR efficiently.Previous reports found that it can be used to detect and regulate CTL by non-covalently linked HLA-A2/Ig G dimers, which is easily dissociated in an acidic environment. Therefore, our group constructed covalently linked AFP/HLA-A2 dimer by two(G4S)3 linkers, the dimer still could not tolerate the SPA acid elution buffer. To solve the problem, this study covalently links AFP peptide with ?2m by a(G4S)3 linker, and then binds AFP-(G4S)3-?2m to the ? chain by Fos-Jun leucine zipper structure, which links two peptide chains with non-covalently hydrophobic force. It can load the antigen peptide to HLA-A2 monomer, two such monomer will form p MHC-Ig dimer though disulfide bonds of the Fc part. We hope the dimeric fusion protein can tolerate the SPA in order to obtain high purity intact conformation dimer,which is able to improve the affinity between p MHC dimer with TCR, thereby generate efficiently p MHC-specific alloreactive T cells. The main contents and results are as the following:1. Preparation of AFP/HLA-A2 dimer linked by Fos-Jun The jun, fos sequence and Ig G1-Fc gene fragments were cloned from p Fast BacTMDual+[DR15/Ig G1-Fc]plasmid which was constructed in our previous study. In addition, AFP-(G4S)3-?2m and HLA-A2(?1-?3) gene fragments were cloned from p MD19-T + AFP-(G4S)3-?2m plasmid and T2 cell, respectively. Next, Overlap-PCR was used to recombine the c DNA of AFP-(G4S)3-?2m region with Jun sequence and HLA-A2 region with Fos sequence. Then, chimeric p Fast BacTMDual+[AFP-(G4S)3-?2m-Jun]+[HLA-A2-Fos/Ig G1-Fc]plasmid was constructed by inserting AFP-(G4S)3-?2m-Jun, HLA-A2-Fos, Ig G1-Fc into the downsteam of the p Fast BacTMDual plasmid promoters, Ph and P10, respectively. Restriction enzyme digestion and specific primer PCR analysis results showed that p Fast BacTMDual +[ AFP-(G4S)3-?2m-Jun ] + [ HLA-A2-Fos/Ig G1-Fc ]recombinant plasmid contained AFP-(G4S)3-?2m, HLA-A2 and Ig G1-Fc fragments. Sequence analysis demonstrated the p Fast BacTMDual + [ AFP-(G4S)3-?2m-Jun ] +[HLA-A2-Fos/Ig G1-Fc]chimeric gene was correctly constructed as expected. p Fast BacTMDual+[AFP-(G4S)3-?2m-Jun]+[HLA-A2-Fos/Ig G1-Fc]recombinant plasmid and DH10 BacTM helper plasmid were co-trasformated into Baculovirus shuttle plasmid to form Bacmid+[AFP-(G4S)3-?2m-Jun]+[HLA-A2-Fos/Ig G1-Fc] plasmid, which was transfected into SF9 insect cells using Cellfectin Reagent. The supernatant contained recombinant virus and the fusion protein, which was reused to infect new SF9 cells. The fusion protein in the supernatant was purified by using SPA, and identified by ELISA, Western blotting and flow cytometry. We detected abundant AFP/HLA-A2 fusion protein in the purified SF9 cells supernatant, and peaking at fifth generation after infection by ELISA. Western blotting identified the molecular weight of the AFP/HLA-A2 monomer about 77 KD, which was confirmed to our expectation. Moreover, flow cytometry result showed that the AFP/HLA-A2 dimer combined to mononuclear cells from HLA-A2 negative individuals with some affinity.2. Conformation stability analysis of AFP/HLA-A2 dimer linked by Fos-Jun and Covalently linked AFP/HLA-A2 dimer Our previous study had made the covalently linked AFP/HLA-A2 dimer in CHO cells, but the structure of the dimer was decomposed for that the ?2m fragment was easy to tall off in the low p H eluent(0.1 M glycine p H 3.0) when cell supernatant was purification by protein A chromatographic column. In this experiment, we covalently linked AFP peptide with ?2m by(G4S)3, and then linked the a chain with AFP-(G4S)3-?2m by leucine zipper structure, which overcame the influence of the acid environment and guaranteed intact structure of the AFP/HLA-A2 dimer. Double sandwich ELISA verified that the AFP/HLA-A2 dimer linked by Fos-Jun had an increased recovery efficiency comparing to the previously dimer.In brief, we successfully established the preparation and purification methods of AFP/HLA-A2 dimer linked by Fos-Jun, which is proved boosting p MHC restrictive alloreactive T cells. The p MHC restricted CTLs provided a solid foundation for further study of adoptive immunity cell therapy for tumor.