| A variety of immune cells and molecules (membrane and soluble) involves in immune responses, a complicated and strictly regulated progress for organisms. B7-CD28 family of ligands and receptors defined co-stimulatory and inhibitory pathways that have crucial roles in the modulation of T lymphocyte cells activation, immunological tolerance and T cells responses. Recently, Watanabe and colleagues have identified an inhibitory Ig superfamily receptor, BTLA (B and T lymphocyte attenuator) and its ligand, B7x (B7S1;B7-H4), which is a new member of the B7 superfamily. Although BTLA shares relatively low sequence homology (9-13% identity) with other receptors for B7 family members, it is structurally similar to CTLA-4 and PD-1. BTLA is a type I transmembrane glycoprotein with an extracellular single IgV-like domain, a transmembrane region and a cytoplasmic region. Indeed, a single IgV-like domain is a characteristic feature among all receptors for B7 family members. Additionally, the BTLA cytoplasmic region contains three tyrosine residues, which are contained within sequence motifs: a Grb2 potential binding site, an immunoreceptor tyrosine inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). These motifs are conserved in the murine and human orthologs and are features shared with PD-1. ITIM motif exsists among inhibitory receptors and can bind and activate tyrosine phosphatase. The activated typosine phosphatases cuase the dephosphorylation of the phosphorylated tyrosines, thus it refrain some cells activation that depends on protein-tyrosine kinase. ITIM motif plays a key role in inhibitory immune response. The presence of an ITIM sequence suggests that BTLA might function as an inhibitory receptor. After T-cell differentiation, BTLA is expressed only on Th1 cells and its expression is independent on interleukin-12 (IL-12) or interferon-γ (IFN-γ) signaling, suggesting a specific role of BTLA in Thl cells. At the same time, BTLA transcripts can also be detected in primary B cells and B-cell lines, suggesting that this molecule could also regulate B-cell responses. In vitro, polarized BTLA-deficient Th1 cells show enhanced proliferation, compared with wild-type Th1 cells. When BTLA-deficient mouse wasimmuned with NK-KLH, the level of IgGl, IgG2a and IgG2b rised 3-folds respectively, comparing with control. BTLA-deficient mice showed enhanced disease incidence and severity in a model of experimental autoimmune encephalomyelitis (EAE). The treatment with anti-B7x blocking antibodies exacerbated EAE, whereas B7x-Ig infusions inhibitted T-cell proliferation. Moreover, B7x-Ig treatment inhibitted CD8"cytotoxic T lymphocyte (CTL) maturation and proliferation, and prolonged experimental animals survival in a graft-versus-host disease (GVHD) model. The regulation of singals transduction between ligands and receptors is available for tumor rejection, induction of immunological tolerance, precaution and treatment of autoimmune, hypersensitivity and allogenic graft rejection. Just like CTLA-4 and PD-1, BTLA should have the similar effect in the T lymphpcyte cells inhibitory response and matainance of peripheral tolerance.Based on previous analysis, immune response may be regulated by blocking the mutual function between BTLA and B7x, referring to the study of CTLA-4 ,PD-1 and their ligands. In this study, exBTLA gene fragment was amplified by reverse transcription -polymerase chain reaction (RT-PCR) and ligated into vector pSec/WG to prepare the recombinant plasmid exBTLA/pSec/WG, which would express the target gene effectively in eukaryotic cells. Following sequence verification, the identified recombinant plasmids were transformed into Flp-in CHO cell lines with Lipofectamine 2000 reagent. The culture medium of these recombinant cells was pooled and the target protein was purified with Protein G-Sepharose 4B column.The contents and results of this research were as follows:Firstly, we design primers with DNAstar software according to exBTLA gene sequence. The recombinant plasmid exBTLA/pSec/WG was prepared by ligating exBTLA gene which was prepared by RT-PCR, and the vector pSec/WG expressing in mammals with high efficiency. The correctness of recombinant was identified by double enzyme digestion and sequencing.Secondly, the identified recombinant plasmids were transformed into Flp-in CHO cell lines with Lipofectamine 2000 reagent. After selection with hygromycin in culture medium, recombinant cell clones expressing the target gene were obtained.Thirdly, the culture medium of the recombinant cells was pooled and the target protein was isolated with Protein G-Sepharose 4B Fast Flow column. The purity of the isolatedfusion protein was identified with sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) method and the size of the isolated protein was checked with Western-blot assay. Resutls showed that high-purity protein with the same molecular size of exBTLA-IgG Fc was obtained. The protein samples were condensed with PEG 20000 and the protein concentration was measured with BCA method.Fourthly, Balb/c mice were immunized with exBTLA-IgG4 Fc fusion protein and monoclonal antibodies against exBTLA were prepared with hybridoma method. Three clones belong to IgGl subclass and kappa type were screened out and named as 2D10, 1C3 and 1A6 respectively, Balb/c mice were primed with pristine and then inoculated with well-grown hybridomas intraperitoneally. The induced ascites were proved containing anti-human exBTLA monoclonal antibody with enzyme-linked immunoadsordent assay (ELISA) and their titer reached about 103'—106 respectively; Monoclonal antibody affinity assay showed that the affinity for the three clones were as follows: 2D1O>1A6>1C3; Western blot identification sugested that Mab could bind specially to the antigen.Through the above experiments, we obtained the inhibitory exBTLA-IgG4 Fc fusion protein and purified the target protein with high-purity and the correct molecular size. Three monoclonal antidies against exBTLA protein were obtained and characterized finally. The results provide concrete experimental data for further exploring the role of BTLA in immune homeostasis, studying the controlling factors for expression of inhibitive receptors on naive and activated lymphocyte cells, recognization of accurate target sites of inhibitive receptors within cells, evaluating the sensitivity and biological function of the gene polymorphism of inhibitive receptor in diseases. |
