| Background:The major cause of graft failure for all types of organ transplantation is rejection. Despite an ever-growing armamentarium of immunosuppressive drugs to combat the recipient's immune response to the transplanted organ ,suppression of the allospecific T cell Response remains a major challenge. T cell activation involves binding of the T cell receptor(TCR) to the MHC antigen complex,and an addition interaction between co-stimulatory molecules expressed on the T cell and the antigen-presenting cell(APC). ICOS/ICOSL, CD28/B7 are the two important pairs of co-stimulatory molecules,and biological effects are synergic .Induction of antigen specific hyporesponsiveness and even tolerance in organ transplant recipients is the foundamental way for resolving the problem of toxicity and infection in lifelong antirejection therapy with non-specific immunosuppression. In recent years, a novel strategy that modifies the grafts, with some immunomodulatory genes has been developed, which renders the organ grafts hyporeponsed or tolerated by the recipients with either little side effects or little risk of systemic immunosuppression via ex vivo genetic manipulation. Promising results have been obtained in some animal transplantation model . Thus , the novel strategy offers exciting perspectives for the development of safe and effective immunotherapy in clinical organ transplantation.In my research, the recombinant ICOS extracellular domain protein and CTLA4Ig fusion protein, which are believed to effectively block of B7-CD28 and ICOS-ICOSL costimulatory pathway in T cell acivation, was chosen as the immunomodulator. In our previous research, the recombinant CTLA4Ig adenovious vector was successfully constructed by Docter Huang Chibing. Here, I focused on constructing ICOS extracellular domain gene-recombinant adenovirus . The effective way of transferring these target genes into the renal allografts with gene-recombinant adenovirus was explored. Furthermore, the effect of modification of renal allografts with target genes on the survival of renal allograft was investigated in rats. The main results and conclusions were as follows:Objective: 1. Amplification of ICOS extracellular domain cDNA fragment from tonsil by nest RT-PCR2. Construction and identification of ICOS extracellular domain gene recombinant replication-deficient adenovirus.3. The effective way of recombinant adenovirus transfecting ICOS,CTLA4Ig gene into kidneys of donor rats4. The effect of the survival of renal allografts transfected with ICOS,CTLA4Ig gene Methods and Results:1. Amplification of ICOS extracellular domain cDNA fragment from tonsil by nest RT-PCRIn this study, with the help of computer-aided analysis, two pairs of PCR primers were designed based on the published ICOS cDNA in Genebank which encoding 1-149aa of the extracellular domain and a signal peptide. A 470bp cDNA fragment was amplified by reverse-transcription methods followed by two cycles of PCR from the total RNA of human tonsil。Moreover, the fragment was then verified by sequencing analysis to make sure that the cDNA is just the one we expected. 2. Construction and identification of ICOS extracellular domain gene recombinant replication-deficient adenovirus.In this research, we use AdEasy system from Stratagene Company to construct ICOS recombinant replication-deficient adenovirus. Firstly, we clone ICOS extracellular domain gene into an AdEasy transfer vector —pAdtrack-cmv plasmid; Secondly, the resulting plasmid pAdtrack-cmv-ICOS was then linearized with pmeI and co-transformed into the E.coli strain BJ5183 together with pAdEasy-1, the viral DNA plasmid. The pAdEasy-1 is E1 and E3 deleted; its E1 functions can be complemented in 293 cells. Recombinants are selected with kanamycin and screened by restriction enzyme analysis. The recombinant adenoviral construct is then cleaved with PacI to expose its ITR (Inverted Terminal Repeats) and transfected into 293 cells to produce viral particles. The resulting recombinant adenovirus was identified by PCR... |
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