Example 3
To confirm the introduction of the HSV1-TKmut gene expression in OTS-412, wild type vaccinia virus and OTS-412 were identified by restriction enzyme mapping. After respectively infecting the wild-type vaccinia virus and OTS-412 into human osteosarcoma cells, the viruses were isolated and viral genomic DNAs were extracted to obtain a negative control (Wild type-VV) and a positive control (OTS-412).
The obtained viral DNAs were digested with HindIII restriction enzyme (10 units/2.5 μg) and separated by size using a DNA electrophoresis apparatus (FIG. 5). As a result, when comparing the negative control group and the positive control group, four corresponding bands (arrows) and one mismatching band (dotted arrow) between 4 kb and 8 kb were identified. The mismatching band had a large gene size, which showed that the HSV1-TKmut gene and the firefly luciferase gene were inserted into the TK region of vaccinia virus. It was confirmed as a unique band pattern of OTS-412 different from that of the wild-type vaccinia virus. When the wild-type vaccinia virus and OTS-412 after several passages were compared with the control groups, the same band patterns as those of the respective control groups were observed, confirming that the HSV1-TKmut gene in OTS-412 had genetic stability.
