The Study Of The Foundation And Evalution Of Recombinated Vector MB_7.1-pcDNA₃

Tumor disease is the most harmful to peoples' health.Recently,with the development of molecular biology and new comprehension of the reasons and mechanisms of tumor, gene carcinoma immune therapy has become a hot method today.When normal cells become the carcino cells,there are large changes on the construction,biostructure and metabolism of the carcino cells.The changing carbohydrate protein would be the new antigen of the carcino cells which includes tumor associated antigen(TAA) and tumor specific antigen(TSA).TSA only lies on the the surface of the carcino cells.Not only the active immunity but also the adoptive immunity were used in antitumor therapy ,it would encounter the activating and multiplying of T cell.Activation of T lymphocytes is an essential event for the efficient response of the immune system. Optimal T cell activation requires at least two stimuli. A primary stimulus is generated upon the interaction of a foreign antigen presented by an APC in the context of MHC class I or class II molecules, with the TcR; this specific interaction between antigen and TCR is imited by the class I and class II molecules with CD8 and CD4. A secondary stimulus is delivered through the interaction of costimutatory molecules on the antigen-presenting cells (APCs) with receptor present on T lymphocytes. Interaction of the costimulatory B7 molecules on antigen presenting cells with CD28 on T lymphocytes appears to be particularly important, as it modifies secretion of cytokines, especially interleukin-2. In the absence of CD28 costimulation, T cells enter a state of anergy that can be overcome by interleukin-2 (IL-2), but not subsequent costimulation. These different interactions initiate a series of signals that are transduced from the cell membrane to the nucleus through different specific signaling pathways that regulate cytokine gene transcription. An immediate consequence of triggering the comlpex of T-cell antigen receptor (TCR) and CD3 is activationg of cellular tyrosine kinases (PTKs) including P56lck,P59fyn and ZAP-70 which result in activation of downstream signals pathway such as PLC-γ,PI3K,Ras/MAPK,Itk.CD28,Interact-ing with B7,triggers costimulatory signals critical for the T-cell response,which signals into the cell through different biochemical pathway that involve IP3K,Pyk2,Vav1,PKC-θand A-Smase.The B7 molecules can be expressed on APCs including monocytes, macrophages, B cells, dendritic cells,and activated T cells. Despite the fact that many tumor express MHC class I molecules presenting"foreign"peptide antigens,avigorous tumor-destructing immune response is seldom detected.A possible explanation is that rumor cannot provide adequate costimulatory signals as provided by professional antigen presenting cells (APCs). The stimulation of a specific antitumor immune response, involving the recruitment of T cells and induction of T-cell effector functions, is an attractive possibility for cancer immunotherapy. In the past few years, advances in our understanding of the mechanisms of T-cell activation and costimulation have provided the basis for strategies to enhance antitumor immunity and break tolerance. These strategies include the equipment of tumor cells with costimulatory molecules such as B7, blockade of inhibitory signals on T cells (e.g.through cytotoxic T-lymphocyte antigen 4,CTLA) and grafting of T cells with antigen-triggered, recombinant costimulatory receptors. Combining antigen triggered activation with appropriate costimulatory pathways will lead to novel approaches to improve the efficacy of T-cell-mediated adoptive immunotherapy of malignant diseases.In this study ,Mouse B7-1 cDNA was cloned by RT-PCR from BALB/C mouse splenic cells and inserted into pcDNA3 to construct an eukaryotic expression vector. This constructor was named mB7.1- pcDNA3, in which the B7-1 cDNA was identified by digestion (with EcoRⅠand XhoⅠ) and sequencing to be consistent with the data from Genbank. mB7.1- pcDNA3 plasmid was tr...