Construction, Expression And Identification Of Chimeric HLA-A2/H-2K~b-Fc Fusion Protein

Major histocompatibility complex (MHC) encodes a group of antigens which can cause a strong and rapid graft rejection when these antigens are mismatched in donor and recipient. MHC molecules have important biological significance in immune response. On the one hand, MHC molecules are directly involved in endogenous or exogenous antigen handling and processing; On the other hand, in the TCR recognition of the antigenic peptide presented by APC, the peptide must be associated with the MHC, ie, the ligand of TCR is peptide/MHC complex (pMHC). Therefore, MHC is important in T cell recognition of antigen.The MHC classⅠmolecule consists of two polypeptide chains, one is heavy chain orαchain, which includesα1,α2 andα3 domains; another is the light chain orβ2-microglobulin (β2m). The antigen-binding groove is composed ofα1 andα2 domains with antigenic peptide resident in. Theα3 domain has a highly conserved sequence, and it has the homology to the constant region of Ig. This domain is able to be associated with CD8, a cell surface glycoprotein found in CTL. Theβ2m is combined with theα3 domain via non-covalent bond, which is necessary for the MHC class I molecule to maintain the stability of natural structure and molecular expression.HLA-A2 is a common allele of human MHC classⅠmolecule, which accounts for 40%~60% HLA-A alleles nearly in all populations, therefore, HLA-A2 is chosen in this study. The mouse MHC is H-2, the class I of it comprises H-2K, H-2D and H-2L loci. We have been interested in animal model for in vivo transplantation studies, the HLA-A2 transgenic mouse provides us with a good animal mode. On the other hand, the polymerized pMHC (eg, pMHC tetramer and pMHC dimer) is able to bind to antigen specific T cells with higher avidity, which has been widely applied in the detection for the antigen-specific T cells. When the HLA-A2 transgenic mouse and the HLA-A2 tetramer or dimer are used to study HLA-A2-restricted T-cell response, question arising is that theα3 domain of HLA-A2 would not interact with the mouse CD8 efficiently, and cause the detection of antigen-specific T cells inefficient. The aim of this study is to construct and expression HLA-A2/H-2K~b-Fc dimmer, this dimer is composed of HLA-A2α1,α2 domains, H-2K~bα3 domain, mouseβ2m, and IgG2a Fc fragment. Theα3 domain and theβ2mof HLA-A2 dimer is replaced by the mouse counterparts, which is expected to more efficiently interact with the HLA-A2 restricted T cells generated in the transgenic mouse.The main points of this study are described as following:1. Construction of HLA-A2/H-2K~b-Fc hybrid geneTheβ2m, HLA-A2α1-α2 domain, H-2K~bα3 region and IgG2a-Fc gene fragments are cloned from the mRNA of C57 mice spleen cells, pcDNA3.1 (+) [HLA-A2/Fc] plasmid, pET28 (a) [H-2K~b] plasmid and pcDNA3.1 (+) [H-2Kd / IgG2a - Fc] plasmid, respectively. The chimeric HLA-A2/H-2K~b gene was constructed by overlap-PCR to recombine the cDNA of HLA-A2α1-α2 region with H-2K~bα3 region. Then, cDNA ofβ2m, HLA-A2/H-2K~b and IgG2a-Fc were cloned into pFastBacTMDual plasmid which has double promoters, to form pFastBacTMDual- [HLA-A2/H-2K~bFc&β2m] recombinant plasmid. Restriction enzyme digestion and specific PCR analysis results showed pFastBacTMDual[HLA-A2/H-2K~bFc&β2m] recombinant plasmid containβ2m, HLA-A2/H-2K~b and Fc fragments. Sequence analysis demonstrated theβ2m-HLA-A2/H-2K~b-IgG2a-Fc chimeric gene was correctly constructed.The pFastBacTMDual[HLA-A2/H-2K~bFc&β2m] recombinant plasmid was transformed into DH10BacTM E. coli which contains the Bacmid plasmid, by the supporting of the helper plasmid transposition function in trans, the chimeric gene recombined into baculovirus genome to form a large recombinant plasmid. Specific PCR confirmed that target gene was inserted into plasmid Bacmid successfully.2. Expression and identification of HLA-A2/H-2K~b-Fc fusion proteinThe pFastBacTMDual[HLA-A2/H-2K~bFc&β2m] plasmid was transfected into sf9 insect cells using Cellfectin Reagent. The supernatant contains recombinant virus and the fusion protein, which can be reused to infect new sf9 cells. The fusion protein in the supernatant was purified by using SPA, and identified by ELISA, Westernblotting and flow cytometry, the results confirm that the protein had the correct molecular weight and conformation.In this study, we constructed an HLA-A2/H-2K~bFc fusion protein, which is expected to effectively bind to antigen-specific CD8+T cells of the HLA-A2 transgenic mice. Moreover, the dimeric form of the pMHC molecules could largely increase the affinity between the dimer and T cells compared to the monomer. Therefore, construction and expression of the HLA-A2/H-2K~b-Fc fusion protein will provide a basis to research the mechanisms and interventions of acute graft rejection in HLA-A2 transgenic mouse model.