Construction and identification of recombinant adenovirus Ad5-hNK4-EGFP

Objective To construct recombinant adenovirus Ad5-hNK4-EGFP with enhance green fluorescent protein(EGFP) tag and NK4 gene and observe its effect on the proliferation of breast cancer MDA-MB-231 cells.Methods The hNK4 gene was amplified with high fidelity DNA polymerase using plasmid pcDNA3 / hNK4 as a template,and cloned into adenovirus shuttle vector pAdTrack-CMV.The constructed recombinant shuttle plasmid pAdTrack-hNK4 was linearized with PmeⅠ and inserted into adenovirus backbone plasmid pAdEasy-1 for homologous recombination in E.coli BL5183.The obtained recombinant adenovirus plasmid pAd-hNK4-EGFP was linearized with PacⅠ,then transfected to AD-293 cells for packaging.The obtained recombinant adenovirus Ad5-hNK4-EGFP was subjected to four cycles of propagation and determined for titer.AD-293 cells were infected with the recombinant adenovirus and determined for transcription of NK4 gene and expression of NK4 protein by RT-PCR and Western blot respectively.The effect of Ad5-hNK4-EGFP on proliferation of MDA-MB-231 cells induced by hepatocyte growth factor(HGF) was determined by MTT method.Results Digestion with PacⅠ proved that recombinant adenovirus plasmid pAd5-hNK4-EGFP was constructed correctly.After packaging and four cycles of propagation,recombinant adenovirus Ad5-hNK4-EGFP reached a titer of 1.5 × 1011 PFU / ml.RT-PCR and Western blot showed that the NK4 gene in Ad5-hNK4-EGFP was expressed in AD-293 cells.HGF promoted the proliferation of MDA-MB-231 cells,while Ad5-hNK4-EGFP antagonized the proliferation induced by HGF.Conclusion Recombinant adenovirus Ad5-hNK4-EGFP for expression of NK4 gene was successfully constructed,which antagonized the proliferation of MDA-MB-231 cells induced by HGF.It laid a foundation of further study on the functions,interaction and action mechanisms of HGF and NK4 and provided a novel target for gene therapy of breast cancer.