| Objective To construct tumor-specific replication-defective adenovirus with CAIX promoter and the controlled hrGFP-1 reporter. To lay the foundation for the detection of circulating tumor cells in peripheral blood.MethodsUsing double enzymatic digestion(BglII and PmeI), hrGFP-1 was obtained from pShuttle-CMV-IRES-hrGFP-1 and added to pShuttle-CAIX-E1 A. After ligation using T4 DNA ligase, the mixture were transformed into bacteria DH5α. The correctly constructed recombinant shuttle plasmid pShuttle-CAIX-E1A-IERS-hrGFP-1 was linearized undergoing PmeI enzymatic digestion and transformed to competent BJ5183 including plasmid pAdEasy-1. Through this, Ad vector was generated by homologous recombination. The acquired adenovirus plasmid pAd5-CAIX-E1A-IRES-hrGFP-1 was digested with PacI and transfected to HEK293 cells for packaging. The obtained recombinant adenovirus Ad5-CAIX-E1A-IRES-hrGFP-1 was purified and measured for titer after four cycles of amplification.Results Recombinant shuttle plasmid and adenovirus plasmid were constructed correctly and the virus were packaged successfully. Following enzymatic digestion and PCR, recombinant plasmid received gel electrophoresis. The results were in consistency with the analysis in Genebank and Software Vector NTI Advance 11 which confirmed right insertion of hrGFP-1 and successful homologous recombination in BJ5183. During packaging and the amplification of virus, the fluorescent protein were expressed steadily and cytopathic effect was observed. The titer of recombinant adenovirus after four cycles of amplification was 1.87×1010pfu/ml.Conclusion Recombinant adenovirus Ad5-CAIX-E1A-IRES-hrGFP-1 was constructed successfully which indicated the possibility of application into clinical stages and genetic levels. With this less invasive and repeatable biomarker(CTC), to detect metastasis invasion, assess prognosis and monitor tumor response against various types of therapies become viable. |
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