Construction And Identification Of Coxsackie Adenovirus Receptor-independent Tumor-specific Replication-defective Recombinant Adenovirus In Kidney

Objective To construct coxsackie adenovirus receptor-independent tumor-specific replication-defective recombinant adenovirus in kidney with carbonic anhydrase IX promoter and humanized renilla reniformis green fluorescent protein-1reporter. To establish a new method for the detection of circulating tumor cells in peripheral blood of kidney cancer patients and provide a theoretical basis for clinical treatment.Methods The hrGFP-1 target gene and the vector fragment are obtained from pShuttle-CMV-hrGFP-1 and pShuttle-CAIX-E1 A by using double enzymatic digestion(BglII and PmeI), and connected using T4 DNA ligase overnight to construct recombinant shuttle plasmid pShuttle-CAIX-E1A-hrGFP-1. Nextly, shake and strain plasmid. The correctly constructed recombinant shuttle plasmid pShuttle-CAIX-E1A-hr GFP-1 confirmed by PCR was linearized with PmeI digestion and transformed to competent BJ5183 with plasmid AdEasy-1 / f11 p successively. To construct recombinant adenoviral vector pAd5 / f11p-CAIX-E1A-hr GFP-1 by homologous recombination. The recombinant adenovirus plasmid had been constructed correctly confirmed by PCR and restriction enzyme digestion analysis was transformed into competent DH5 a bacteria to amplify and save. The acquired recombinant adenovirus plasmid pAd5/f11p-CAIX-E1A-hrGFP-1 was digested with PacI and transfected to HEK293 cells for packaging. The obtained recombinant adenovirus Ad5/f11p-CAIX-E1A-hrGFP-1 was purified and measured for titer after four cycles of amplification.Results Recombinant shuttle adenovirus plasmid pAd5/f11-CAIX-E1A-hr GFP-1 was constructed correctly as proved by PCR and restriction analysis. The titer of recombinant adenovirus after four cycles of amplification and purification was 1×108 pfu/ml.Conclusion The coxsackie adenovirus receptor-independent kidney tumor-specific replication-defective recombinant adenovirus Ad5/f11-CAIX-E1A-hr GFP-1 was constructed successfully. Providing a new method for early detection of the kidney cancer patients circulating tumor cells in peripheral blood has important significance on the early detection of malignant invasion ofkidney cancer, staging, prognosis, individual treatment and evaluation of the treatment.