Construction And Identification Of Carrying Humanized Green Fluorescent Protein Bladder Cancer Tissue-specific Adenoviral Vectors

Objective Carrying humanized green fluorescent protein(hr GFP) bladder cancer tissue-specific adenoviral vectors pAd5-PSCAE-UPII-E1A-IRES-hrGFP was uesed for the detection of circμlating tumor cells in peripheral blood. For the detection of bladder cancer circμlating tumor cells provides a new detection methodMethods According to the map, BglII and PmeI were chosen to enzyme pShuttle-CMV-IRES-hrGFP and pShuttle-PSCAE-UPII-E1 A.Afer the electrophoresis,The target fragments of PSCAE-UPII-E1 A and IRES-hrGFP was recovered by Agarose Gel DNA Extraction Kit.T4 ligase was used to connect Two target fragment at 16 ° incubator overnight.Ligation mixture was transformed into DH5α competent cells; plated, positive clones were selected after 16 hours, overnight shaking bacteria,PCR and DNA sequencing appraisaled the new Shuttle pShuttle-PSCAE-UPII-E1A-IRES-hr GFP, the new Shuttle pShuttle-PSCAE-UPII-E1A-IRES-hr GFP is constructed. The pShuttle-PSCAE-UPII-E1A-IRES-hr GFP was linearized by Pme I and transformed into the competent cell of BJ5183(containing pAdEasy vector) by Electroporation.plated, positive clones were selected after 16 hours, overnight shaking bacteria, The recombinant vector pAd-PSCAE-UPII-E1A-IRES-hr GFP was appraisaled by PCR and enzyme(BspH1and Pac I).pAd-PSCAE-UPII-E1A-IRES-hrGFP was transfected into HEK293 cells to package adenovirus.Results BglII and PmeI were chosen to enzyme pShuttle-CMV-IRES-hrGFP and pShuttle-PSCAE-UPII-E1 A. T4 ligase was used to connect Two target fragment the new Shuttle pShuttle-PSCAE-UPII-E1A-IRES-hrGFP is constructed. PCR and DNA sequencing appraisaled the new Shuttle pShuttle-PSCAE-UPII-E1A-IRES-hr GFP, the recombinant vector pAd5-PSCAE-UPII-E1A-IRES-hrGFP constructed by Homologous recombination in bacteria, the recombinant vector pAd-PSCAE-UPII-E1A-IRES-hrGFP were appraisaled by PCR and enzyme. pAd-PSCAE-UPII-E1A-IRES-hr GFP wastransfected into HEK293 cells by Lipofectamine 2000, Fluorescence microscopy observed the expression of hr GFP Gene.The adenovirus was successfμlly packaged.After the adenovirus was purified,Adenovirus titer is 1.87×1011pfu/ml.Conclusion In order to obtain the two target fragments, We used two restriction endonuclease. The two target fragments formed the new Shuttle vector pShuttle-PSCAE-UPII-E1A-IRES-hr GFP by Ligation. The recombinant vector pAd5-PSCAE-UPII-E1A-IRES-hrGFP was successfμlly constructed by homologous recombination.The bladder cancer tissue-specific adenovirus was prepared. The bladder cancer tissue-specific adenovirus provides a basis for the detection of circμlating tumor cells.