A novel vector for a rapid generation of fiber-mutant adenovirus based on one step ligation and quick screening of positive clones.

The generation of fiber-modified adenoviral vector has proven difficult. In the paper, we developed a new system for rapid construction of fiber-modified adenoviral vector containing foreign peptides in the HI loop or C-terminal of the fiber knob. The new system was established through the following processes. First, a unique BamHI mutation was made in the genome of Ad5 without causing amino acid change. Second, two unique restriction enzymes BamHI and SfuI, both with sticky end, were introduced in the HI loop or C-terminal of Ad5 fiber knob. Third, a lacza expression cassette was placed between BamHI and SfuI sites for a quick identification of positive cloning based on white-blue color screening. This system allows generation of recombinant adenoviral vector by a single step, in vitro ligation followed by quick white-color positive clone screening. To prove the principle of the method, Ad5HI-RGD by modifying HI-loop of the fiber knob with RGD motif and Ad5Cter-PK7 by modifying C-terminal of the knob with poly-lysine (pK7) were successfully generated in vitro. Ad5 with a knob modified in the HI loop of the fiber with Tat-PTD, NGR or SIKVAV peptide were also successfully developed. The transduction of the modified viruses for Hela, U87 MG and MDA-MB-231 cells was investigated in vitro compared with unmodified Ad5. In conclusion, the new vector system allows for a rapid generation of fiber-mutant adenovirus and provides useful tool for gene function analysis and cancer gene therapy.