For cloning the pTP gene of the adenoviral genome into adenoviral shuttle vector, pVAX1-3484-CMVp-ΔE1B, total RNA was isolated from cells with standard Trizol/chloroform extraction following 24 h post infection with E1B55K-deleted oncolytic adenovirus. cDNA was synthesized by RT-PCR using random hexamer. Synthesized cDNA was used as template to perform PCR using primers for the full pTP gene obtained from NCBI (NC_001405.1 or AC_000008). Sense/antisense primer sequences are as follows: 5′-ATGGCCTTGAGCGTCAACGATT-3′, 5′-CTAAAAGCGGTGACGCGGG-3′. PCR products were ligated into pMD20-T vector (Takara, USA), which has a dT-overhang at the 3′ end. Then, PCR product in pMD20-T vector was cloned into the expression vector pCDNA3.1-hygro(+) (Invitrogen, Carlsbad, CA, USA) at the BamHI and XbaI sites. To improve expression of the pTP gene, Kozak sequence (GCCACC) was inserted in front of the pTP gene by annealing. Expression of pTP protein was confirmed by western blotting using pTP antibody. Next, the pTP gene with Kozak sequence was subcloned into an adenoviral shuttle vector, pVAX1-3484-CMVp-ΔE1B, at the HindIII and SalI-blunted sites. The adenoviral shuttle vector, pVAX1-3484-CMVp-ΔE1B-Kozak-pTP, was linearized by PmeI digestion. The adenoviral vector dl324-BstBI was linearized by Bsp119I digestion, and the two linearized vectors were co-transformed into E. coli BJ5183 cells for homologous recombination. The following virus amplification step was the same as for other adenoviruses.

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