Construction Of Urothelium-specific Recombinant Adenovirus And Its Inhibition

Background:Gene therapeutic approaches to bladder cancer have shown research promise. Recombinant adenoviruses currently are used for gene therapy.The differential expression of the desired gene in the target tissue is essential for gene therapy.Aim of the study is to construct urothelium-specific recombinant adenovirus and investigate its inhibition in bladder cancer cell.Methods:RT-PCR analysis was used to determine expression patterns of hUPII and Coxsackie adenovirus receptor on multiple cell lines.Transient transfection and luciferase detecting assay were used to detect tissue specificity of the hUPII promoter.E1A fragments were digested from the vector RpS-TOAD-PSE-PBN-E1A.HUPII promoter fragments were amplified from the vector CP1131 with primers.E1 A fragments and hUPII promoter fragments were inserted into pShuttle vector RpS-TOAD to create the vector Rp-UPII-E1A.E1A fragments were cut off from the vector Rp-UPII-E1A to establish the vector Rp-UPII-Null.Rp-UPII-E1A and Rp-UPII-Null were cotransfected with pAdEasy-1 by electroporation in electrocompetent E.coli BJ5183 cells respectively.Positive plasmid was transformed into E.coli DH5αcells for large-scale amplification.Two positive plasmids were transfected into 293 cells by Lipofectamine 2000.After 7 days,viruses were harvested.After replicating to adequate volume in 293 cells,the adenovirus was purified by a routine method with CsCl.Virus titer was determined by the methods of UV-SDS assay and TCID50.Restrictive enzyme digestion assay and PCR confirmed the correct construction.The adenovirus E1 A protein expressed in BIU-87 was tested by Western blot after cells were infected with recombinant adenovirus.Recombinant adenovirus Ad-UPII-E1A was tested for its inhibition in bladder cancer cell line BIU-87.Results:HUPII and CAR were expressed and the hUPII promoter is highly active in bladder cancer cell line BIU-87.Using homologous recombination in bacteria technology,the hUPII promoter and E1A gene were inserted into the genome of type 5 recombinant adenovirus.The E1A protein was markedly positive in the samples of BIU-87 cells infected with recombinant adenovirus Ad-UPII-E1A.MTT assay demonstrated recombinant adenovirus Ad-UPII-E1A inhibited bladder cancer cell BIU-87 growth.Conclusion:The hUPII promoter shows high tissue specificity.Recombinant adenovirus Ad-UPII-E1A and Ad-UPII-Null were constructed and confirmed.Recombinant adenovirus Ad-UPII-E1A is effective in inhibition in bladder cancer cell line BIU-87.