Construction And Identification Of Adenoviral Vector Containing P53 Gene And Flt3 Ligand

Objective:To construct adenoviral vector containing MUC1 promoter and the fusion gene of p53/Flt3L,to express the gene of p53/Flt3L.Give full play to the role of p53 tumor suppressor and Immune therapy of dendritic cells,to induce the apoptosis of breast cancer cells.To further explore the conditions the adenovirus vector provide for the treatment of breast cancer.Methods:Artificial synthetic MUC1 promoter,p53 gene and Flt3 ligand gene,and adding the appropriate restriction sites in the two sides of the sequence,and useing the method of restriction enzymolysis,connection to clone them into pUC19 vector,and pUC19-MUC1,pUC19-p53 and pUC19-Flt3L were obtained,the whole volume of the reaction solution was transformed into the competent cells and extract plasmid DNA and the DNA sequencing was confirmed to be correct.restructure the above three plasmids using the appropriate restriction enzyme,synthesis and extraction the plasmids of pUC19-MUC1-p53 and pUC19-MUC1-Flt3L.p53 gene and Flt3 ligand were amplified by PCR,the fusion gene was constructed by using a linker sequences,the fusion gene was inserted the into plasmid of pUC19-MUC1 plasmid,the plasmid was verified by double digesti-on, agarose gel electrophoresis and the fragment was recovered.The pUC19-MUC1-p53-linker-Flt3L were sequenced.Using the multiple cloning sites, double restriction enzyme to digestion,the DNA fragment of purpose were recovered,phosphorylated and end up flated.The objective gene were inserted into the line of the pAxcwit2 of adenovirus vector,the recombinant plasmid of pAx-MUC1-p53,pAx-MUC1-Flt3L,pAx-MUC1-p53-linker-Flt3L were successfully constructed.Confirm the DNA sequencing and restriction analysis of the three recombinant plasmid and find that the inserted foreign gene were correspond with corresponding gene sequences of gene bank.It demonstrated that recombinant adenoviral vector carrying MUC1 promoter and fusion of p53/Flt3L was successfully constructed.Results:The synthetic MUC1 promoter,p53 gene and Flt3 ligand sequence were confirmed by DNA sequencing that the inserted foreign gene were correspond with corresponding gene sequences of gene bank.The recombinant plasmids of pAx-MUC1-p53,pAx-MUC1-Flt3L,pAx-MUC1-p53-linker-Flt3L were confirmed by the method DNA sequencing,restriction enzyme digestion and agarose gel electrophoresis,the results show that the exogenous gene sequences of recombinant adenovirus vector were consistent with the known sequences.The band length of 15% agarose gel electrophoresis released were consistent with the known gene fragment.It confirmed the synthesis gene were proper correct,adenovirus vectors were successfully constructed.Conclusion:1. The adenovirus vector MUC1 promoter and fusion gene of p53/Flt3L constructed by gene synthesis technology were consistent with the known sequences,that create the necessary conditions for recombinanting DNA.2. Successfully applicat MUC1 as a promoter constructed adenovirus vector,using MUC1 as a promoter to further enhance the targeting of adenovirus vector for the treatment of breast cancer cells in the later experiments.3. Adenovirus vector using MUC1 as promoter and bringing p53/Flt3L fusion gene was successfully constructed.