Construction and identification of replication-deficient adenovirus expressing human Interleukin-21 gene.

Objective To establish a replication-deficient adenovirus expressing human interleukin-21(IL-21)gene and green fluorescent protein and thus provide a basis for further research on the gene therapy by IL-21 gene.Methods Total RNA was extracted from human peripheral blood lymphocytes.IL-21 gene was amplified by RT-PCR.The PCR products and pDC316-mCMV-EGFP vector were digested with endonucleases NotI and HindIII respectively and linked to construct pDC316-IL21-EGFP recombinant vector.pDC316-IL21-EGFP was identified by endonuclease digestion and sequencing.Ad5F35-IL21-EGFP recombinant adenovirus was generated by cotransfecting 293 cells with pDC316-IL21-EGFP and pBHGlox_E1,3CreF35.The titer of Ad5F35-IL21-EGFP was measured.The cell growth curve and cell cycle of EC-9706 cells transfected with Ad5F35-IL21-EGFP were detected.Results The identification of pDC316-IL21-EGFP by endonuclease digestion and sequencing showed that IL-21 gene fragment was recombined into pDC316-mCMV-EGFP vector correctly with the right open reading frame and that DNA sequence of IL-21 gene in the recombinant vector was the same as that in GenBank database.Mature adenovirus was established by packaging Ad5F35-IL21-EGFP into 293 cells.Ad5F35-IL21-EGFP expressed IL-21 gene and green fluorescent protein.The physical titer and infectious titer of Ad5F35-IL21-EGFP were 9×1011 VP/ml and 5×1010 IU/ml,respectively.The growth of EC-9706 cells transfected with Ad5F35-IL21-EGFP was slower than that transfected with control adenovirus(P 0.05).EC-9706 cells of G1 phase increased,and the cells of G2 and S phases decreased.Conclusion IL-21 gene recombinant adenovirus expressing green fluorescent protein is constructed successfully,and the titer of recombinant adenovirus is high.The IL-21 gene recombinant adenovirus can transfect EC-9706 cells efficiently and inhibit EC-9706 cell growth.