Construction and identification of recombinant adenovirus nm23-H1

Objective To construct a recombinant adenovirus encoding for non-metastatic 23rd H1(nm23-H1) gene. Methods Amplified the nm23-H1 gene from template pcDNA3.1-nm23-H1 by PCR technology. Subcloned the gene into the pShuttle-CMV shuttle plasmid. The product pShuttleCMV-nm23-H1 was linearized by Pme I to mediate homologous recombination with pAdEasy-1 in AdEasier-1 host bacteria. The positive clone was identified by enzyme digestion, PCR analysis and DNA sequence analysis. After linearized by Pac I , the recombinant adenovirus DNA pAd-nm23-H1 was transfected into 293 cells for packaging and amplification of Adeno-nm23-H1. Adeno-nm23-H1 was further identified by PCR analysis and DNA sequence analysis. Results PCR analysis, enzyme digestion and DNA sequence analysis proved the pShuttleCMV-nm23-H1 and the pAd-nm23-H1 had been successfully constructed. After being packaged in 293 cells, the recombinant adenovirus Adeno-nm23-H1, whose titer was as high as 4. 3×109 viral particle/ml were produced and further identified by PCR analysis and DNA sequence analysis. No wide type virus was generated. Conclusion We have successfully constructed a recombinant adenovirus Adeno-nm23-H1 which encoding nm23-H1 gene. This virus will be useful for the understanding of nm23-H1gene and research on the potential mechanism of metastasis suppression.