Construction And Identification Of Recombinant Adenoviral Plasmid Expressing The N-terminal Domain Of TIMP-3

Objective TIMP-3 (tissue inhibitor of metalloproteinase-3) plays an important role in the development of pancreatic cancer. However, the effect of N-TIMP3 (N-terminal domain of TIMP-3) on pancreatic cancer is still not clear. The aim of this study was to construct and identify recombinant adenoviral plasmid expressing N-TIMP3, for further investigating the effect of N-TIMP3 on pancreatic cancer. Besides, recombinant adenoviral plasmid expressing EGFP (enhanced green fluorescent protein) was constructed and identified as a positive internal control of the adenoviral plasmid Construction system.Methods Construction of the recombinant shuttle plasmids (pShuttle-N-TIMP3 and pShuttle-EGFP) were carried out by inserting the DNA sequence encoding N-TIMP3 or EGFP into pShuttle-CMV. Subsequently, the recombinant adenoviral plasmids (pAd-N-TIMP3 and pAd-EGFP) were obtained by homologous recombination between the Pme I linearized products of those shuttle plasmids and the adenoviral backbone plasmid pAdEasy-1. And the recombinant adenovirus particles (Ad-N-TIMP3 and Ad-EGFP) were obtained by transfection of QBI-293A cells with Pac I linearized recombinant adenoviral plasmids. The titer of recombinant adenoviruses was determined by TCID₅₀ (tissue culture infectious dose 50) after two additional cycles' proliferation. Finally, PANC-1 cells were infected by either Ad-N-TIMP3 or Ad-EGFP, and the expression of N-TIMP3 was tested by Western blot, and that of EGFP was tested by Inverted Fluorescence Microscope.Results The recombinant shuttle plasmids (pShuttle-N-TIMP3 and pShuttle-EGFP) were identified to be successfully constructed. And the recombinant adenoviral plasmids (pAd-N-TIMP3 and pAd-EGFP) were successfully generated by homologous recombination. After that, the cytopathic effect (CPE) could be observed after transfection of QBI-293A with Pac I linearized recombinant adenoviral plasmids. Ad-N-TIMP3 and Ad-EGFP were titered at 5×10⁸PFU/ml and 8×10⁸PFU/ml respectively after two additional cycles' proliferation. Finally, the expression of N-TIMP3 or EGFP was found in PANC-1 cells infected by either Ad-N-TIMP3 or Ad-EGFP.Conclusions The recombinant adenoviral plasmids (pAd-N-TIMP3 and pAd-EGFP) expressing N-TIMP3 or EGFP were successfully constructed and identified, which would be beneficial for further investigating the effect of N-TIMP on pancreatic cancer, and the possibilities of gene therapy.