The Study Of Knocking MHC-1 Out Of Vascular Endothelial Cell By Intracellular Antibody

Backgroud and Objective:Replacing pathological or depleted organs with able-bodied organs to reconstruct the normal physiological function has been the clinicians' pursuit for a long time.But the reject reaction always happens after transplantation to result in functional incapacitation.Immunosuppressive therapy is a commonly used method to prevent and treat rejection after transplantation.It was well-known including both non-drug treatment and drug treatment.The former reacted in earlier period such as radiation,lymph-drag,splenectomize and local immunity,but they had been scarcely used because of the adverse effects.Conversely, immunosuppressive drug is used universally nowadays.The pulsion of neotype immunosuppressive drugs brings galactic assist to clinical work.But long-term taking drugs can bring many adverse reactions such as infection,malignant tumor and side effects of their own which enormously influence the achievement ratios and the long-term surviving of the transplant.With the development of gene engineering, gene therapy opened up a new therapy to rejection.MHC-I is the main attacking target of allograft.If the expression of MHC-I in the donor cells is suppressed or knocked out,the rejection may be eased or avoided.Therefore,The aims of present study are to construct AAV bearer contains gene of MHC-I intrabody,authenticate the target shooting of the intrabody in the cells by transfected AAV into endothelial cells of allantoic veins,and so as to approach the effect and significance of intrabody in immunological rejection.Methods:1.F105VH-scFv-KDEL cDNA was cloned by PCR and T carrier PCR. Accreditted and sequenced by molecular biologic methods.2.F105VH-scFv-KDEL cDNA was obtained by restriction enzyme and then inserted it into the EcoRI—BamHI site of pSSHG.3.The recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells.4.ECV304 was transducted stably with rAAV,and detected whether F105VH-scFv-KDEL cDNA was conformed successfully in ECV304 by RT-PCR.5.The MHC-I expression was confirmed by immunocytochemistry.6.The survival state was observed by microcytotoxicity.Results:1.F105VH-scFv-KDEL cDNA was accreditted correctly by restriction enzyme,electrophoresis and DNA sequencing.2.F105VH-scFv-KDEL cDNA was inserted into pSSHG successfully confirmed by cleavage map.3.The viral tite was 3.4×10⁹-3.4×10¹⁰PFU/ml.4.F105VH-scFv-KDEL cDNA was conformed in ECV304 successfully.5.The expression of MHC-I.in rAAV group cells was fewer than that in PBS group cells.6.The survival cells of rAAV group were more than those of PBS group.Conclusion:1.F105VH-scFv-KDEL cDNA was successfully cloned.2.pSSHG/F105VH-scFv-KDEL was constructed successfully.3.High density rAAV was packed successfully.4.The rAAV can knock out the MHC-I of ECV304 effectually.