Vaccines based on the use of mv
A virus and kit technology, applied in the field of recombinant modified Ankara vaccinia virus, can solve problems such as insufficient therapeutic effect
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[0089] Materials and methods
[0090] Plasmid construction
[0091] The ubiquitin / tyrosinase fusion gene was constructed and cloned into the MVA transfer vector pIIIdHR-P7.5. Here we establish a hybridization PCR method in which the ubiquitin (Ub) gene can be fused to the tyrosinase (hTyr) cDNA without insertion of any additional non-Ub or non-hTyr DNA sequences. Because ubiquitin expressed as a protein fusion is cleaved by cytoplasmic proteases at its G76 residue, we focused on mutating G76 to A76, which has been shown to inhibit cytoplasmic cleavage when expressed from a plasmid vector (Rodriguez F et al. J Virol, 1997).
[0092] In a first step, ubiquitin was amplified from RNA preparations of murine B16 melanoma cells in standard reverse transcriptase PCR according to the manufacturer's instructions (Titan One Tube RT-PCR System, Roche). Select primer 5′-GGG C GG ATC C GA CCA TGC AGA TCT TCGTGA AGA CCC TGAC-3′ and 5′-CAA AAC AGC CAG GAG CAT CGC ACCTCT CAG GCG A...
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