| Co-Investigator(Kenkyū-buntansha) |
TAKAGI Michihiro Kobe Univ., Fac.of Agr., Inst., 農学部, 助手 (90301283)
SUGIMOTO Chihiro Obihiro Univ.of Agr.and Vet.Med., Res.Center for Protozoan Mol.Immunol., Prof., 原虫病研究センター, 教授 (90231373)
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| Research Abstract |
Marek's disease virus (MDV) causes malignant lymphomas in chickens, but the molecular mechanism of the transformation remains unknown. Strains of MDV are classified into 3 serotypes based on their pathogenicity : only serotype 1 strains of MDV (MDV1), except for attenuated vaccine strains (i.e. CVI988), are oncogenic. Although Marek's disease (MD) is effecivecontrolled by vaccination, the protection mechanism by live vaccines is not known, and emergence of highly virulent MDV which can cause vaccine breaks has been reported. For this reason, a recombinant MDV which can induce tumor-specific apoptosis was constructed for the development of a newly invented anti-tumor vaccine in this study. In addition, differencein the structure of the meq gene has been identified between oncogenic and attenuated MDV1. Since the meq gene is known as an MDV oncogene, detailedanalysis on the change in the viral genome was done to obtain the insight into the role of the meq gene on the transformation by MD
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A gene cassette to express an apoptosis-inducing gene, the VP3 gene cloned from chicken infectious anemia virus, under the control of the meq promoter was constructed. The meq promoter was chosen since the meq gene has been known to be expressed only in tumor or MDV latently-infectedcells. When this gene cassette was introduced into an MD tumor-derived cell line, MSB1, viability of MSB1 was decreased within 24 hours, and DNA fragmentation was also detected in total cellular DNA prepared from MSB1. However, no change in the cell viability was observed in chick embryo fibroblast (CEF) transfected with this cassette. These results suggest that this gene cassette could be used for the tumor-specific induction of apoptosis.
To study genetic differences between oncogenic and nononcogenic MDV1, polymerase chain reaction (PCR) was performed to amplify the meq gene in the viral genome. In addition to a PCR product including the native meq open reading frame (ORF), a 1.2-kb product was amplified from the DNA sample prepared from CEF infected with CVI988, but not with oncogenic strains, RB1B and Md5. Sequence analysis showed that a 178-bp sequence was inserted to the meq ORF of CVI988 (L-meq gene). This L-meq gene was also detected in an oncogenic MDV1, strain JM, which had been passaged for more than 70 times in vitro. Since this insertion could potentially cause a frame shift in the meq ORF, the resultant ORF could encode for the Meq protein with a different transacti vator domain. The L-meq gene was also detected in the MDV genome prepared from MD tumor-derived cell lines. The biological significance and function of this long meq gene is not yet elucidated, but this insertion may also contribute to the changes in the biological properties of different MDV1 strains. Less
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