Construction And Identification Of Adenovirus Vectors Containing Recombinant Human Coagulation Factor Ⅷ Gene

The genetic defect of human coagulation factor VIII gene will cause the disorder of coagulation system. This single gene disorder known as hemophilia. Aiming at the shortcomings of the current method of hemophilia A alternative treatment, try to construct a vector for gene therapy for hemophilia A. Study on construction of recombinant virus vector system based on Ad Easy, Firstly, using molecular cloning methods, obtained the sequence of coagulation factor VIII gene of human. And the gene fragment was sequenced to verify the obtained analysis, Cloned into the shuttle plasmid vector pShuttle. To monitor the recombinant hf VIII gene expression, the Aequorea victoria green fluorescent protein gene coding sequence used into the shuttle plasmid vector. The GFP and hf VIII genes were respectively cloned into the downstream and upstream of the IRES region under the control of a CMV promoter. The incorporation of GFP encoding sequence in this vector was designed to serve as a live marker for convenient assessment of adenovirus tansducing efficiency. By constructing the expression vector containing CMV promoter,Mediated coagulation factor VIII gene and green fluorescent protein gene transcription and translation are common in subsequent cells. The recombinant plasmid containing GFP-FVIII fragments and adenovirus genomic DNA p Ad Easy-01 vector to complete recombination in Escherichia coli strains. After ligation, the conversion reaction, recombinant plasmid was extracted by alkaline lyses method, enzyme digestion, agarose gel electrophoresis analysis and restriction enzyme analysis. And ultimately obtained the adenoviral vector p Ad-hF Ⅷcontaining factor Ⅷgene. The plasmid vector was constructed on the basis of success, the p Ad-h F vector was linearized by Ⅷrestriction enzyme PacI, and then use liposomal transfection method, cultured virus packaged in HEK 293 cells. Seven to ten days after viral culture cells, intracellular virus initial liquid molecular detection and analysis, access to the positive DNA bands containing the target gene. Meanwhile, the use of Western blot hybridization technique, in a recombinant viral vector infection by cultures grown cells, clotting factors h F Ⅷdetected immunological activity. This study obtained recombinant human coagulation factor Ⅷgene vectors for application of gene therapy and for reference materials of hemophilia A.